Respiratory syncytial virus: vaccines

ABSTRACT

Polypeptides, nucleotides, and compositions useful for preparing diagnostic reagents for and vaccines against human Respiratory Syncytial Virus are disclosed. The polypeptides include short polypeptides which are related to a neutralizing and fusion epitope of the Respiratory Syncytial Virus fusion protein or a neutralizing epitope of the G protein.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.07/102,180, filed Sep. 29, 1987, currently abandoned.

TABLE OF CONTENTS

1. Field of the Invention

2. Background of the Invention

2.1 Respiratory Syncytial Virus in Disease

2.2 Immunological Approach to the Prevention of RS Virus Infection

2.3 Recombinant DNA Technology and Gene Expression

2.3.1. E. coli As An Expression Vector

2.3.2. Vaccinia Virus As An Expression Vector

2.3.3. Baculovirus As An Expression Vector

3. Summary of the Invention

4. Brief description of the Figures

5. Detailed Description of the Invention

5.1 Identification of a Neutralizing and/or Fusion Epitope[s] Of RSVirus Fusion Protein

1.1.1 Mapping by Defined Proteolytic Cleavage

5.1.2 Cloning and Expressing Fragments of the Fusion Protein

5.1.3 Synthesis of Antigenic Peptides

5.2 Preparation of Proteins, Polypeptides and Peptides Related to RSVirus Fusion Protein and G Protein

5.3 Insertion of the RS Virus Fusion Protein or G Protein CodingSequences into Expression Vectors

5.4 Identification of Recombinant Expression Vectors Capable ofReplicating and Expressing the Inserted Gene

5.5 Identification and Purification of the Expressed Gene Product

5.6 Determination of the Immunopotency of the Recombinant Product

5.7 Formulation of a Vaccine

5.7.1 Subunit Vaccine Formulations

5.7.2 Viral Vaccine Formulations

5.7.3 Passive Immunity and Anti-Idiotypic Antibodies

5.8 Diagnostic Assays

5.8.1 Immunoassays

5.8.2 Nucleic Acid Hybridization Assay

6. Protection of Animals: RS Virus Fusion Protein

6.1 General Procedures

6.1.1 Isolation of Fusion Protein

6.1.2 Assays

6.1.2.1 Virus Neutralization Assay

6.1.2.2 Anti-Fusion Assay

6.1.2.3 Enzyme Immunoassay (EIA)

6.2 Protection of Animals: Homologous and Heterologous Protection

6.3 Protection of Baboons

6.4. Protection Against Bovine RS Virus

6.5. Avoidance of Enhanced Disease

7. Protection of Humans: RS Virus Fusion Protein

8. Identification of Neutralizing and/or Fusion Epitopes[s] of RS VirusFusion Protein

8.1 General Procedures

8.1.1 Protein Immunoblot (Western Blot Analysis)

8.1.2 Coupling of Peptides to Keyhole Lympet Hemocyanin (KLH) andProduction of Rabbit Antisera

8.1.3 Proteolytic Cleavage of Fusion Protein

8.1.4 Dot Blot Analysis

8.2 Mapping by Defined Proteolytic Cleavage

8.3 Mapping by Expression of Protein Fragments

8.4 Mapping by Synthetic Peptides

9. Immunogenicity of Modified RS Virus Fusion Protein

9.1. Conformational Modification

9.2. Deacylated Fusion Protein

10. Protection of Animals: RS Virus G Protein

10.1. Isolation of G Protein

10.2. Passive Protection

10.3. Active Immunization and Protection

11. Immunogenicity of RS Virus G Protein Expressed in RecombinantVectors

12. Cell-Mediated Immunological Aspects of RS Virus Vaccine

13. Deposit of Microorganisms

1. FIELD OF THE INVENTION

Respiratory Syncytial (RS) virus is a major cause of lower respiratorydisease in infancy and early childhood. It is a matter of great medicaland scientific interest to provide safe and effective vaccines againstthis virus.

RS virus is an enveloped RNA virus. The major outer envelope proteins,the F protein (also known as the fusion protein or fusion glycoprotein)and the G protein, play a key role in RS viral infection becauseantibodies directed against these proteins can neutralize the virus.

For the purposes of this Application, a neutralizing epitope on a viralprotein is an epitope which is essential for virus infectivity asdefined by the fact that antibody binding to the epitope neutralizes thevirus. Likewise, a fusion epitope on a viral protein is an epitope whichis essential for virus-cell fusion or intercellular spread of infectionby fusion between an infected-cell and an uninfected cell as defined bythe fact that antibody-binding to the epitope abrogates fusion.

The present invention relates to compositions and methods of preparationof proteins and polypeptides associated with the outer envelope of RSvirus. More particularly, one embodiment of the invention is directed tocompositions and methods for preparation of proteins and polypeptidesrelated to the fusion protein of RS virus. The proteins and polypeptidesof this embodiment of the invention are related to a neutralizingepitope, or a fusion epitope, or both, of the fusion protein, and may beused as immunogens in vaccine formulations including multivalentvaccines for active immunization and for the generation of antibodiesfor use in passive immunization, as well as reagents for diagnosticassays.

Another embodiment of the invention is directed to compositions andmethods for preparation of proteins and polypeptides related to the Gprotein of RS virus. The proteins and polypeptides of this embodiment ofthe invention are related to a neutralizing epitope of the G protein,and may be used as immunogens in vaccine formulations includingmultivalent vaccines for active immunization and for the generation ofantibodies for use in passive immunization, as well as reagents fordiagnostic assays.

The novel proteins and polypeptides related to a neutralizing epitope, afusion epitope, or both can be obtained by using either recombinant DNAor chemical synthetic methods. Additionally, the invention relates tonovel DNA sequences and vectors useful for expressing RS virus relatedproteins and polypeptides and to cells which harbor the novel DNAsequences and vectors.

It should be noted that an epitope is a three dimensional structuregenerated by the molecular arrangement of an underlying molecularentity. In the present invention, the underlying molecular entities arepolypeptides. It is well known that the structural properties ofpolypeptides of which three dimensional configuration is one, may onlybe minutely changed by the introduction of a small number ofmodifications such as substitutions, insertions and deletions of one ormore amino acids. Generally, such substitutions in the amino acidsequence of a polypeptide are in the amount of less than twenty percent,more usually less than ten percent. Generally, conservativesubstitutions are less likely to make significant structural changesthan non-conservative substitutions, which in turn are less likely tomake significant structural changes than insertions or deletions.Examples of conservative substitutions are glycine for alanine; valinefor isoleucine; aspartic acid for glutamic acid; asparagine forglutamine; serine for threonine; lysine for arginine; phenylalanine forthreonine; and the converse of the above. Therefore, it is to beunderstood that the present invention embraces modified polypeptides solong as the epitope is unchanged.

It is also well known that viral epitopes may exhibit strain-to-strainvariations. Adjustment by the above-indicated modifications may indeedbe used advantageously.

Finally, the fusion protein and G protein related polypeptides orproteins of the invention, like the bonafide viral proteins, may belabeled or unlabeled, bound to a surface, conjugated to a carrier, andthe like, depending on the use to which they are put.

BACKGROUND OF THE INVENTION 2.1. Respiratory Syncytial Virus in Disease

RS virus is a major cause of lower respiratory disease in infancy andearly childhood (McIntosh and Chanock, 1985, in Virology, Fields, B.(ed), Raven, NY, pp. 1285-1304). In all geographical areas, it is themajor cause of bronchiolitis and pneumonia in infants and youngchildren. The agent reinfects frequently during childhood, but illnessproduced by reinfection is generally milder than that associated withthe initial infection and rarely causes major problems.

RS virus is an enveloped RNA virus of the family Paramyxoviridae and ofthe genus pneumovirus. The two major envelope proteins are the Gprotein, which is responsible for attachment of the virus to the hostcell membrane, and the fusion protein, which is responsible for fusingthe virus and cell membranes. Virus-cell fusion is a necessary step forinfection. Fusion protein is also required for cell-cell fusion which isanother way to spread the infection from an infected cell to anuninfected cell.

Antibodies directed against the fusion protein or against the G proteincan neutralize the virus. However, only antibodies to the fusion proteinwill block the spread of the virus between cells, i.e. have anti-fusionactivity. Thus, antibodies to the fusion protein will protect againstcirculating virus as well as inhibit the spread, between cells, of anestablished infection. Antibodies to the fusion protein (both polyclonalantisera against purified fusion protein and monoclonal antibodies whichcontain both neutralizing and anti-fusion activity) have been found tobe protective in animal models against infection (Walsh et al., 1984,Infect. Immun. 43:756-758).

2.2. Immunological Approach to the Prevention of Virus Infection

A practical means for protection of infants and young children againstupper and lower respiratory disease would be protective vaccinationagainst RS virus. Vaccination of expectant mothers (active immunization)would protect young children by passive transfer of immunity, eithertransplacentally, or through the mother's milk. Several approaches to anRS virus vaccine are possible, but some of them have proven unsuccessfulin the past.

Vaccination with killed RS virus vaccine has been tried and found to beineffective (Kim et al., 1969, Am. J. Epid. 89:422). Not only werechildren not protected, but in some cases, subsequent infections with RSvirus resulted in atypical and more severe disease than in theunimmunized controls. This phenomenon is not unique to RS virus and hasbeen seen also in killed paramyxovirus vaccines such as measles. It hasbeen suggested that the reason for the failure of the past inactivatedRS virus vaccine was due to inactivation of the biologically functionalepitopes on either or both of the viral envelope glycoproteins. That isto say, the neutralizing and fusion epitopes on the killed virus vaccinewere "denatured". As a result, the vaccinated subject did not experiencethe biologically functional neutralizing and fusion epitopes. Therefore,when the vaccinated subject encountered a live virus, the resultantantibody response did not yield protective immunity. Instead, there wasan antibody mediated inflammatory response which often resulted in amore severe disease (Choppin and Scheid, 1980, Rev. Inf. Dis., 2:40-61).

The second approach to an RS virus vaccine has been to attenuate livevirus. Temperature sensitive mutants (Wright et al., 1982, Infect.Immun. 37:397-400) and passage attenuated virus (Belshe et al., 1982, J.Inf. Dis. 145:311-319) have proven to be poorly infectious and notefficacious in the prevention of disease when used as immunogens in RSvirus vaccines. However, in these cases, there was no atypical diseaseas a result of vaccination.

Based on our current knowledge of the structure of RS virus and theimmune response to infection, it is clear that a useful vaccine to thisvirus must be effective in inducing production of antibodies to thefusion protein and/or the G protein. Of particular importance toprotective immunity is the production of antibodies that inhibit fusionand therefore, can stop the spread of virus between cells in therespiratory tract. Additionally, it may be helpful to induce a cellmediated immune response, including the stimulation of cytotoxic T cells(CTL's) which are useful against RS virus infected cells. The variousvaccine formulations of the present invention are directed to meetingboth these objectives.

2.3. Recombinant DNA Technology and Gene Expression

Recombinant DNA technology involves insertion of specific DNA sequencesinto a DNA vehicle (vector) to form a recombinant DNA molecule which iscapable of being replicated in a host cell. Generally, but notnecessarily, the inserted DNA sequence is foreign to the recipient DNAvehicle, i.e., the inserted DNA sequence and DNA vector are derived fromorganisms which do not exchange genetic information which in nature, orthe inserted DNA sequence comprises information which may be wholly orpartially artificial. Several general methods have been developed whichenable construction of recombinant DNA molecules. For example, U.S. Pat.No. 4,237,224 to Cohen and Boyer describes production of suchrecombinant plasmids using processes of cleavage of DNA with restrictionenzymes and joining the DNA pieces by known methods of ligation.

These recombinant plasmids are then introduced by means oftransformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture.Because of the general applicability of the techniques describedtherein, U.S. Pat. No. 4,237,224 is hereby incorporated by referenceinto the present specification. Another method for introducingrecombinant DNA molecules into unicellular organisms is described byCollins and Hohn in U.S. Pat. No. 4,304,863 which is also incorporatedherein by reference. This method utilizes a packaging, transductionsystem with bacteriophage vectors (cosmids).

DNA sequences may also be inserted into viruses, for example, vacciniavirus. Such recombinant viruses may be generated, for example, bytransfection of plasmids into cells infected with virus (Chakrabarti etal., 1985, Mol. Cell. Biol. 5:3403-3409).

Regardless of the method used for construction, the recombinant DNAmolecule is preferably compatible with the host cell, i.e., capable ofbeing replicated in the host cell either as part of the host chromosomesor as an extra-chromosomal element. The recombinant DNA molecule orrecombinant virus preferably has a marker function which allows theselection of the desired recombinant DNA molecule(s) or virus(es). Inaddition, if all of the proper replication, transcription andtranslation signals are correctly arranged on the recombinant DNAmolecule, the foreign gene will be properly expressed in the transformedor transfected host cells.

Different genetic signals and processing events control gene expressionat different levels. For instance, DNA transcription is one level, andmessenger RNA (mRNA) translation is another. Transcription of DNA isdependent upon the presence of a promoter which is a DNA sequence thatdirects the binding of RNA polymerase and thereby promotes RNAsynthesis. The DNA sequences of eucaryotic promoters differ from thoseof procaryotic promoters. Furthermore, eucaryotic promoters andaccompanying genetic signals may not be recognized in or may notfunction in a procaryotic system.

Similarly, translation of mRNA in procaryotes depends upon the presenceof the proper procaryotic signals which differ from those of eucaryotes.Efficient translation of mRNA in procaryotes requires a ribosome bindingsite called the Shine-Dalgarno (SD) sequence on the mRNA. For a reviewon maximizing gene expression, see Roberts and Lauer, 1979, Methods inEnzymology 68:473.

Many other factors complicate the expression of foreign genes inprocaryotes even after the proper signals are inserted and appropriatelypositioned. One such factor is the presence of an active proteolyticsystem in E. coli and other bacteria. This protein-degrading systemappears to destroy foreign proteins selectively. A tremendous utility,therefore, would be afforded by the development of a means to protecteucaryotic proteins expressed in bacteria from proteolytic degradation.One strategy is to construct hybrid genes in which the foreign sequenceis ligated in phase (i.e., in the correct reading frame) with aprocaryotic structural gene. Expression of this hybrid gene results in arecombinant protein product (a protein that is a hybrid of procaryoticand foreign amino acid sequences).

Similar considerations of gene expression in eukaryotic systems havebeen discussed in Enhancers & Eukaryotic Gene Expression, Gluzman &Shenk (Eds.), Cold Spring Harbor Laboratories, Cold Spring Harbor, NewYork 1983, and Eukaryotic Viral Vectors, Gluzman (Ed.), Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y. 1982.

Successful expression of a cloned gene requires efficient transcriptionof DNA, translation of the mRNA and in some instances post-translationalmodification of the protein. Expression vectors have been developed toincrease protein production from the cloned gene. In expression vectors,the cloned gene is often placed next to a strong promoter which iscontrollable so that transcription can be turned on when necessary.Cells can be grown to a high density and then the promoter can beinduced to increase the number of transcripts. These, if efficientlytranslated will result in high yields of protein. This is an especiallyvaluable system if the foreign protein is deleterious to the host cell.

Several recombinant DNA expression systems are described below for thepurpose of illustration only, and these examples should not be construedto limit the scope of the present invention.

2.3.1. E. Coli As an Expression Vector

Many E. coli plasmids are known and have been used to express foreigngenes. For economic reasons, it would be highly preferable to be able toobtain a high level of expression. One way to obtain large amounts of agiven gene product is to clone a gene on a plasmid which has a very highcopy number within the bacterial cell. By increasing the number ofcopies of a particular gene, mRNA levels would normally also increase,which in turn would lead to increased production of the desired protein.

2.3.2. Vaccinia Virus as an Expression Vector

Vaccinia virus may be used as a cloning and expression vector. The viruscontains a linear double-stranded DNA genome of approximately 187 kbpairs and replicates within the cytoplasm of infected cells. Theseviruses contain a complete transcriptional enzyme system (includingcapping, methylating and polyadenylating enzymes) within the virus core.This system is necessary for virus infectivity because vaccinia virustranscriptional regulatory sequences (promoters) allow for initiation oftranscription by vaccinia RNA polymerase, but not by cellular RNApolymerase.

Expression of foreign DNA in recombinant viruses requires the fusion ofvaccinia promoters to protein coding sequences of the foreign gene.Plasmid vectors, also called insertion vectors have been constructed toinsert the chimeric gene into vaccinia virus. One type of insertionvector comprises: (1) a vaccinia virus promoter including thetranscriptional initiation site; (2) several unique restrictionendonuclease cloning sites downstream from the transcriptional startsite for insertion of foreign DNA fragments; (3) nonessential vacciniavirus DNA (such as the thymidine kinase gene) flanking the promoter andcloning sites which direct insertion of the chimeric gene into thehomologous nonessential region of the virus genome; and (4) a bacterialorigin of replication and antibiotic resistance marker for replicationand selection in E. coli. Examples of such vectors are described byMacKett (1984, J. Virol. 49:857-864).

Recombinant viruses are produced by transfection of recombinantbacterial insertion vectors containing the foreign gene into cellsinfected with vaccinia virus. Homologous recombination takes placewithin the infected cells and results in the insertion of the foreigngene into the viral genome. Immunological techniques, DNA plaquehybridization, or genetic selection can be used to identify and isolatethe desired recombinant virus. These vaccinia recombinants retain thefunctions essential for infectivity and can be constructed toaccommodate up to approximately 35 kb of foreign DNA.

Expression of a foreign gene can be detected by enzymatic orimmunological assays (e.g., immunoprecipitation, enzyme-linkedimmunosorbent assay (ELISA), radio-immunoassay, or immunoblotting).Additionally, naturally occurring membrane glycoproteins produced fromrecombinant vaccinia infected cells are glycosylated and may betransported to the cell surface. High expression levels can be obtainedby using strong promoters or cloning multiple copies of a single gene.

2.3.3. Baculovirus as an Expression Vector

A baculovirus, such as Autographica californica nuclear polyhedris virus(AcNPV) has also been used as a cloning or expression vector. Theinfectious form of AcNPV is normally found in a viral occlusion. Thisstructure is largely composed of polyhedrin polypeptide in which virusparticles are embedded. Polyhederin gene expression occurs very late inthe infection cycle, after mature virus particles are formed. Therefore,polyhedrin gene expression is a dispensable function, i.e., non-occludedvirus particles produced in the absence of polyhedrin gene expressionare fully active and are capable of infecting cells in culture.According to European Patent Application Serial No. 84105841.5 by Smithet al., a recombinant baculovirus expression vector can be prepared intwo steps. First, baculovirus DNA is cleaved to produce a fragmentcomprising a polyhedrin gene or a portion thereof, which fragment isinserted into a cloning vehicle. The gene to be expressed is alsoinserted into the cloning vehicle; and it is so inserted that it isunder control of the polyhedrin gene promoter. This recombinant moleculeis called a recombinant transfer vector. Normally, the recombinanttransfer vector in amplified in appropriate host cells. Second, therecombinant transfer vector formed in this way is mixed with baculovirushelper DNA and used to transfect insect cells in culture to effectrecombination and incorporation of the cloned gene at the polyhedringene locus of the baculovirus genome. The resultant recombinantbaculovirus is used to infect susceptible insects or cultured insectcells.

3. SUMMARY OF THE INVENTION

The present invention is directed to polypeptides and proteins relatedto a neutralizing epitope, a fusion epitope, or both, of respiratorysyncytial (RS) virus glycoproteins, including the fusion protein and Gprotein, as well as molecularly cloned gene or gene fragments encodingsuch polypeptides and proteins.

One embodiment of the present invention is directed to polypeptides andproteins related to a neutralizing epitope, a fusion epitope, or both,of the fusion protein of respiratory RS virus as well as molecularlycloned genes or gene fragments, which encode these polypeptides orproteins. Another embodiment of the present invention is directed topolypeptides and proteins related to a neutralizing epitope of the Gprotein of RS virus as well as molecularly cloned genes or genefragments, which encode these polypeptides or proteins. The polypeptidesor proteins of the present invention may be used as immunogens insubunit vaccine formulations for RS virus or as reagents in diagnosticimmunoassays for RS virus. The polypeptides or proteins of the presentinvention may be produced using recombinant DNA techniques orsynthesized by chemical methods.

The present invention is also directed to methods for the molecularcloning of and expression of genes or gene fragments encoding aneutralizing or a fusion epitope, or both, of RS virus fusion protein ora neutralizing epitope of the G protein. Accordingly, the invention isalso directed to the construction of novel polynucleotide sequences andtheir insertion into vectors, including both RNA and DNA vectors, toform recombinant molecules or viruses which can be used to directexpression of polypeptides or proteins related to the epitope(s) in theappropriate host cells. The vectors include plasmid DNA, viral DNA,human viruses, animal viruses, insect viruses and bacterial phages.

Recombinant viruses or extracts of cells which comprise the polypeptidesor proteins of the present invention can also be used as immunogens inviral vaccine formulations. Since a fusion or neutralizing epitope of avirus will be recognized as "foreign" in the host animal, humoral andcell mediated immune responses directed against the epitope(s) will beinduced. In a properly prepared vaccine formulation, this should protectthe host against subsequent RS virus infection.

The polypeptides and proteins of the present invention can also be usedas reagents in immunoassays such as ELISA tests and radioimmunoassays todetect RS virus infections in blood samples, body fluids, tissues, andso forth.

The polynucleotide sequences of the present invention can also be usedas reagents in nucleic acid hybridization assays to detect RS virus inblood samples, body fluids, tissues, and so

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the complete nucleotide and amino acid sequences of the RSvirus fusion protein, reproduced from Collins et al., 1985, Proc. Nat'lAcad. Sci. USA, 81:7683-7687.

FIG. 2 shows the electrophoretic behavior in SDS-polyacrylamide gel of 1ug of the purified RS virus fusion protein under various conditions. Inlane 2, the protein was reduced with 5% beta-mercaptoethanol and heatedto 100° C. prior to electrophoresis; in lane 3, the protein was heatedto 100° C. prior to electrophoresis; in lane 4, the protein was notheated or reduced prior to electrophoresis. Lane 1 contains standardmarker proteins whose molecular weights are indicated in the leftmargin. The right margin shows the molecular weight of the variousfusion protein components. The gel was stained with silver forvisualization.

FIG. 3 shows the positions of synthetic polypeptides 1, 2, 3, 4, and 5(sp1-sp5) ) on a linear map of the F₁ subunit. The F₁ subunitencompasses amino acids 137-574. FIG. 3 also shows the exact amino acidsequence of the synthetic polypeptides.

FIG. 4 shows the positions of proteolytic fragments of the fusionprotein on a linear map of the F₁ subunit.

FIG. 5 is a diagramatic representation of an E. coli recombinant vectorcontaining the complete nucleotide sequence of the RS virus fusionprotein gene.

FIG. 6 shows the amino and carboxy terminal sequences of severalrecombinant proteins expressed in E. coli.

FIG. 7 shows the positions of the recombinant proteins on a linear mapof the F₁ subunit. The fragments shown in shaded bars were reactive withthe L4 monoclonal antibody and those shown in solid bars werenon-reactive with the L4 monoclonal antibody.

FIG. 8 illustrates a dot blot autoradiogram of the four (4) syntheticpolypeptides defined by the chains of amino acids shown in the leftmargin. Lanes 1-4 contain 20 ug, 15 ug, 10 ug and 5 ug of peptiderespectively. Lane 5 contains a positive fusion protein control. Theblot was reacted with the L4 monoclonal antibody and then with ¹²⁵I-protein A.

FIG. 9 is a diagramatic representation of a recombinant expressionvector of pPX1044 containing the complete nucleotide sequence of the RSvirus G protein gene.

DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment of the present invention, a region of the RSvirus fusion protein which defines a neutralizing and fusion epitope[s]has been identified. A method for producing novel proteins, polypeptidesand peptides comprising the epitope[s], and the polynucleotide sequenceswhich encode such novel protein and polypeptides are provided.

The fusion protein of RS virus has an apparent molecular weight of70,000 daltons by sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE). The primary (cistronic) translation productconsisting of 574 amino acid residues is synthesized and subsequentlyprocessed by enzymatic cleavage at a trypsin sensitive site yielding twosubunits designated F₁ (amino acids 137-574) and F₂ (amino acids 1-136).

The complete nucleotide sequence of the fusion protein gene from the A2strain of RS virus (Collins et al., 1984, Proc. Nat 1 Acad. Sci. USA,81:7683-7687) which encodes the fusion protein of 574 amino acids isillustrated in FIG. 1. The numbering system depicted in FIG. 1 is usedthroughout this application. The F₁ (apparent molecular weight 48,000dalton) and F₂ (apparent molecular weight 23,000 dalton) subunits arelinked through a disulfide bond to form a protein which is designatedF₁,2 (apparent molecular weight about 70,000 daltons). When purifiedfrom RS virus or infected cells, the native fusion protein existspredominantly as a dimer (apparent molecular weight 140,000 daltons).The dimeric form is the most immunogenic form of the RS virus fusionprotein.

FIG. 2 shows the electrophoretic mobility of various fusion proteincomponents using SDS-PAGE. In lane 2, the protein was reduced with 5%beta-mercaptoethanol and heated to 100° C. prior to electrophoresis; inlane 3, the protein was heated to 100° C. prior to electrophoresis; inlane 4, the protein was not heated or reduced prior to electrophoresis.Lane I contains standard marker proteins whose molecular weights areindicated in the left margin. The right margin shows the molecularweight of the various fusion protein components. The gel was stainedwith silver for visualization (Morrissey, 1981, Anal. Biochem.117:307-310).

Active immunization of cotton rats with purified RS virus fusion protein(the 140,000 dalton form) results in the production of antibodies whichare effective in virus neutralization and in preventing fusion (see, forexample, Section 6.2, infra). As demonstrated in Section 6.2, thisimmunization protected the lung and nasal tissues from infection by RSvirus. Similar results have been obtained in baboons (see, Section 6.3,infra). Additionally, active immunization of animals with purified RSvirus fusion protein previously reduced with beta-mercaptoethanolprotects animals from subsequent RS virus infection (see Section 6.2,infra).

According to another embodiment of the present invention, substantiallypure polypeptides and proteins related to a neutralizing epitope of theRS virus G protein are provided. The RS virus G protein has an apparentmolecular weight of about 84,000-90,000 daltons and is highlyglycosylated. The nucleotide sequence of the gene encoding the RS virusG protein has been disclosed (Satake et al., 1985, Nucleic Acid Res.13:7795-812; Wertz et al., 1985, Proc. Nat'l Acad. Sci. USA 82:4075-79 .The entire gene sequence encoding the RS G protein has been cloned andexpressed in a recombinant expression vector (see Section 9, infra).Surprisingly, active immunization of animals with a recombinantnon-glycosylated RS virus G protein induced a protective immune response(see Section 10, infra).

A monoclonal antibody to the fusion protein, designated L4 (Walsh andHruska, 1983, J. Virol. 47:171-177), is capable of both neutralizing RSvirus and inhibiting fusion. Therefore, it appears that this antibodyreacts with an epitope on the fusion protein which is essential to bothinfectivity and the fusion function of the fusion protein, i.e., thisantibody reacts with both a fusion epitope and a neutralizing epitope.Moreover, passive transfer of the L4 monoclonal antibody will protectcotton rats from virus infection in their lungs (Walsh et al., 1984,Infect. Immun., 43:756-758).

In addition to the L4 type antifusion, neutralizing epitope, there isyet another antifusion, neutralizing epitope of the fusion protein whichmay be conformationally dependent. This epitope is recognized bymonoclonal antibody A5 (Walsh et al., 1986, J. Gen. Virol. 67:505-513).The reactivity of this antibody appears to be dependent upon the nativeconformation of the fusion protein. When the conformation of the fusionprotein is altered either by heat treatment or by heat treatment and thereduction of disulfide bonds, the A5 type epitope is destroyed. Thepresent studies have examined the role of L4 type and A5 type epitopesof the RS virus fusion protein on the formulation of the protein as aneffective vaccine. Results from a competitive ELISA indicate that onethird of the fusion protein specific antibody in human adults recoveringfrom natural RS infection, is directed against epitopes recognized byeither L4 or A5. Vaccination with purified F protein produces a responsewhich can be 80% blocked by a combination of L4 and A5. Competitivebinding to the F protein, shows that these epitopes overlap by 40%.Based on these studies, it appears that the functional antifusion,neutralizing epitopes can be classified into two categories, L4 type andA5 type.

5.1. Identification of Neutralizing and/or Fusion Epitope[S] of VirusFusion Protein

According to the present invention, the region of the RS virus fusionprotein which is an epitope responsible for eliciting both neutralizingand antifusion antibodies has now been determined. This region has beendefined by three methods. The first method employs defined proteolyticcleavage of the native protein. The second method relates to cloning andexpressing fragments of the fusion protein gene, for example, in E.coli. The third relates to synthesis of synthetic polypeptides whichbind to neutralizing and antifusion antibodies. In all three methodsreactivity with the L4 monoclonal antibody was used to identify desiredfragments. Any other monoclonal antibody which is capable ofneutralizing and preventing fusion of RS virus may be used.

5.1.1. Mapping by Defined Proteolytic Cleavage

The L4 monoclonal antibody was tested for its ability to bind to thefusion protein subunits (F₁ and F₂) by protein immunoblot (Western blot)analysis. Using Western blot as described in Section 8.1, infra, the L4monoclonal antibody was able to bind only to the F₁ subunit.Additionally, the L4 monoclonal antibody has been shown to bind toserotype B virus (Anderson et al., 1985, J. Inf. Dis 151:623-33) definedby the prototype virus designated strain 18537.

In order to map the F₁ subunit, synthetic polypeptides are preparedwhich correspond to various regions along the F₁ subunit. Thesesynthetic polypeptides are coupled to a carrier protein, such as keyholelympet hemocyanin (KLH), and then used separately to immunize rabbits(see Section 8, infra). In the particular example exemplified in Section8.2., five antisera (anti-sp1 through anti-sp5) were obtained. Eachantiserum produced in the rabbits reacted with the uncoupled syntheticpolypeptide corresponding to the immunogen which induced the antiserum,i.e., anti-sp1 reacted with sp1; anti-sp2 reacted with sp2, etc. Allfive antisera reacted with the F₁ subunit of the fusion protein.

The purified fusion protein is then subjected to proteolytic cleavageunder a variety of conditions (see Section 8, infra). In the exampleillustrated in Section 8.2, infra, the proteases used were (1) trypsinwhich specifically cleaves after lysine and arginine residues, (2)endoproteinase Arg-C which cleaves specifically after arginine residues,and (3) endoproteinase Lys-C which cleaves specifically after lysineresidues. Cleavage after a residue means the breaking of a peptide bondat the carboxyl end of the residue. It should be understood that othercombinations of proteases can be used. Therefore, the presentlyexemplified combination should not be construed as a limitation on thepresent invention.

Cleavages are also performed in the presence and absence of the L4monoclonal antibody. The cleaved protein fragments are separated bySDS-PAGE and the cleavage products analyzed by Western blot analysis forthe ability to bind to the L4 monoclonal antibody as well as theanti-synthetic polypeptide antibodies (See an exemplary illustrationSection 8.2, infra). The positions of the proteolytic fragments withinthe fusion protein sequence are deduced from the reactivities of thesecleavage fragments with each of the anti-synthetic polypeptide antisera.For example, as illustrated in Section 8.2., a cleavage fragmentgenerated by trypsin digestion which fragment reacted with anti-sp1would be expected to comprise amino acids 155-169. Furthermore, thefragment must have arginine or lysine at its carboxyl end and aN-terminal residue which follows a lysine or arginine in the sequence ofFIG. 1. Finally, the molecular weight of a fragment can be determined byits mobility in SDS-PAGE. This set of information uniquely predicts thatthe 28,000 dalton fragment generated by trypsin digestion and reactivewith anti-sp1 spans amino acids 137-394 of the F₁ polypeptide. Thepositions of the other cleavage fragments are similarly deduced.

The relationship between the positions of the cleavage fragments and thereactivities of these fragments to the L₄ monoclonal antibody areanalyzed. From the example presented in Section 8.2., it can readily beseen that amino acids 283-327 define the region which is common to allof the L4 positive fragments and absent from all of the L4 negativefragments. This region, therefore, comprises a virus neutralizing and/orantifusion epitope of the RS virus defined by the L4 monoclonalantibody.

5.1.2. Cloning and Expressing Fragments of the Fusion Protein

The cDNA illustrated in FIG. 1 containing the complete nucleotidesequence of the fusion protein gene is cloned into a cloning vector suchas the E. coli plasmid vector pBR322. Due to the degeneracy of thenucleotide coding sequences, other DNA sequences which encodesubstantially the same amino acid sequence as depicted in FIG. 1 may beused. These include but are not limited to nucleotide sequencescomprising all or portions of the fusion protein nucleotide sequencesdepicted in FIG. 1 which are altered by the substitution of differentcodons that encode the same or a functionally equivalent amino acidresidue within the sequence (for example, an amino acid of the samepolarity) thus producing a silent change.

Regions of the fusion protein gene are excised from the cloning vectorby restriction endonuclease digestion and ligated into a compatibleexpression vector (see infra). In the experimental example described inSection 8.2., infra, the E. coli expression vector pUC19 (Yanish-Perronet al., 1985, Gene 33:103-19) was used. The expressed recombinantproteins are screened for reactivity first with polyclonal rabbitantiserum to native fusion protein to identify recombinant fragments andthen with L4 monoclonal to identify those fragments comprising theneutralizing and antifusion epitope. As illustrated in Section 8.3., theRS virus fusion protein sequence common to all of the recombinantproteins reactive with the L4 monoclonal antibody is defined by aminoacid residues 253-298.

5.1.3. Synthesis of Antigenic Peptides

In order to confirm the identify of the neutralizing and antifusionepitope identified as described above, synthetic polypeptides can beprepared corresponding in particular to amino acid residues 299-315;294-315; 289-315; and 283-315 of the RS virus fusion protein. Thesepeptides are analyzed for reactivity with L4 monoclonal antibody. Asillustrated in Section 8.4., infra, polypeptides containing residues294-315; 289-315; and 283-315 react positively with L4; whereas peptide299-315 does not. This indicates that a neutralizing and fusion epitoperesides between residues 283-298 and may be as small as residues294-299.

5.2. Preparation of Proteins, Polypeptides and Peptides Related to RSVirus Fusion Protein and G Protein

The proteins, polypeptides and peptides of the present invention can beprepared in a wide variety of ways. The polypeptides, because of theirrelatively short size may be synthetized in solution or on a solidsupport in accordance with conventional techniques. Various automaticsynthesizers are commercially available and can be used in accordancewith known protocols. See, for example, Stewart and Young, 1984, SolidPhase Peptide Synthesis, 2d Ed., Pierce Chemical Co. The structuralproperties of polypeptides, of which three dimensional configuration isone, may only be minutely changed by the introduction of a small numberof modifications such as substitutions, insertions and deletions of oneor more amino acids. Generally, such substitutions in the amino acidsequence of a polypeptide are in the amount of less than twenty percent,more usually less than ten percent. Generally, conservativesubstitutions are less likely to make significant structural changesthan non-conservative substitutions, which in turn are less likely tomake significant structural changes than insertions or deletions.Examples of conservative substitutions are glycine for alanine; valinefor isoleucine; aspartic acid for glutamic acid; asparagine forglutamine; serine for threonine; lysine for arginine; phenylalanine forthreonine; and the converse of the above. Therefore, it is to beunderstood that the present invention embraces modified polypeptides solong as the epitope of the RS virus fusion protein remains unchanged.

It is also well known that viral epitopes may exhibit strain-to-strainvariations. Adjustment by the above-indicated modifications may indeedbe used advantageously.

The polypeptides of the present invention may be employed as labeled orunlabeled compounds depending on their use. By label is intended amoiety which provides, directly or indirectly, a detectable signal.Various labels may be employed, such as radionuclides, enzymes,fluorescers, chemiluminescers, enzyme substrates, cofactors orinhibitors, particles (e.g. magnetic particles), ligands (e.g. biotin)and receptors (e.g. avidin) or the like. In addition, the polypeptidesmay be modified in a variety of ways for binding to a surface e.g.microtiter plate, glass beads, chromatographic surface, e.g. paper,cellulose and the like. The particular manner in which the polypeptidesare joined to another compound or surface is conventional and findsample illustration in the literature. See, for example, U.S. Pat. Nos.4,371,515; 4,487,715 and the patents cited therein.

Alternatively, recombinant DNA technology may be employed to prepare thepolypeptides biosynthetically.

5.3. Insertion of the RS Virus Fusion Protein or G Protein CodingSequences into Expression Vectors

The nucleotide sequence coding for the RS virus fusion protein or aportion thereof or for the RS virus G protein or a portion thereof isinserted into an appropriate expression vector, i.e. a vector whichcontains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence. According to a preferredembodiment of this mode of the invention, nucleotide sequences codingfor a neutralizing and/or fusion epitope of the fusion protein isinserted into an appropriate expression vector. The coding sequence maybe extended at either the 5' and 3' terminus or both termini to extendbiosynthetically the polypeptide while retaining the epitope. Theextension may provied an arm for linking, e.g., to a label (see below),to a carrier or surface. The extension may provide for immunogenicitywhich may other wise be lacking in some of the shorter antigenicpolypeptides of the invention.

A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include but are not limited to mammaliancell cultures such as Chinese hamster ovary cell host cultures, etc.;mammalian cell systems infected with virus (e.g. vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.baculovirus); microorganisms such as yeast containing yeast vectors orbacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA.In one embodiment the expression vector can be an attenuatedenteroinvasive bacteria including but not limited to Salmonella spp.,enteroinvasive E. coli (EIEC), and Shigella spp. Such bacterium caninvade gut epithelial tissue, disseminate throughout thereticuloendothalical system and gain access to mesenteric lymphoidtissue where they multiply and induce humoral and cell-mediatedimmunity. The expression elements of these vectors vary in theirstrength and specificities. Depending on the host-vector systemutilized, any one of a number of suitable transcription and translationelements may be used. For instance, when cloning in mammalian cellsystems, promoters isolated from the genome of mammalian cells, (e.g.,mouse metallothionien promoter) or from viruses that grow in thesecells, (e.g. vaccinia virus 7.5K promoter) may be used. Promotersproduced by recombinant DNA or synthetic techniques may also be used toprovide for transcription of the inserted sequences.

Specific initiation signals are also required for efficient translationof inserted protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where the RS virusfusion protein gene or the RS virus G protein gene including its owninitiation codon and adjacent sequences are inserted into theappropriate expression vectors, no additional translational controlsignals may be needed. However, in cases where only a portion of the RSvirus fusion protein or G protein coding sequence is inserted, exogenoustranslational control signals, including the ATG initiation codon mustbe provided. The initiation codon must furthermore be in phase with thereading frame of the protein coding sequences to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be a variety of origins, both natural andsynthetic.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinations (genetic recombination).

The invention is not limited to the use of E. coli or procaryoticexpression vectors. Expression vectors which can be used include, butare not limited to the following vectors or their derivatives: human oranimal viruses such as vaccinia viruses or adenoviruses; insect virusessuch as baculoviruses; yeast vectors; bacteriophage vectors, and plasmidand cosmid DNA vectors to name but a few.

In cases where an adenovirus is used as an expression vector, the RSvirus fusion protein gene or fragment thereof or the RS virus G proteingene or fragment thereof is ligated to an adenovirustranscriptional/translational control complex, e.g., the late promoterand tripartite leader sequences. This chimeric gene is then inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe RS virus fusion protein or G protein related protein in infectedhosts. Presently, there are two strains of adenovirus (types 4 and 7)approved and used as vaccines for military personnel. They are primecandidates for use as vectors to express the RS virus fusion protein orG protein gene and fragments thereof.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes thechimeric gene product in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers,(e.g., zinc and cadmium ions for metallothionein promoters). Therefore,expression of the genetically engineered RS virus fusion protein or Gprotein or fragment thereof may be controlled. This is important if theprotein product of the cloned foreign gene is lethal to host cells.Furthermore, modifications (e.g., glycosylation) and processing (e.g.,cleavage) of protein products are important for the function of theprotein. Different host cells have characteristic and specificmechanisms for the post-translational processing and modification ofproteins. Appropriate cell lines or host systems can be chosen to ensurethe correct modification an processing of the foreign protein expressed.

5.4. Identification of Recombinant Expression Vectors Capable ofReplicating and Expressing the Inserted Gene

Expression vectors containing foreign gene inserts can be identified bythree general approaches: (a) DNA-DNA hybridization, b) presence orabsence of "marker" gene functions, and (c) expression of insertedsequences. In the first approach, the presence of a foreign geneinserted in an expression vector can be detected by DNA-DNAhybridization using probes comprising sequences that are homologous tothe foreign inserted gene. In the second approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain "marker" gene functions (e.g., thymidinekinase activity, resistance to antibiotics, transformation phenotype,occlusion body formation in baculovirus etc.) caused by the insertion offoreign genes in the vector. For example, if the RSV virus fusionprotein gene or fragment thereof is inserted within the marker genesequence of the vector, recombinants containing the RS virus fusionprotein inserted can be identified by the absence of the marker genefunction. In the third approach, recombinant expression vectors can beidentified by assaying the foreign gene product expressed by therecombinant. Such assays can be based on the physical, immunological, orfunctional properties of the gene product.

Once a particular recombinant DNA molecule is identified and isolated,several methods may be used to propagate it, depending on whether such arecombinant constitutes a self-replicating unit (a replicon). A selfreplicating unit, e.g., plasmids, viruses, cells etc., can multiplyitself in the appropriate cellular environment and growth conditions.Recombinants lacking a self-replicating unit will have to be integratedinto a molecule having such a unit in order to be propagated. Forexample, certain plasmid expression vectors upon introduction into ahost cell need to be integrated into the cellular chromosome to ensurepropagation and stable expression of the recombinant gene. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity.

5.5. Identification and Purification of the Expressed Gene Product

Once a recombinant which expresses the RS virus fusion protein gene orfragment thereof or the RS virus G protein or fragment thereof isidentified, the gene product should be analyzed. This can be achieved byassays based on the physical, immunological or functional properties ofthe product. Immunological analysis is especially important where theultimate goal is to use the gene products or recombinant viruses thatexpress such products in vaccine formulations and/or as antigens indiagnostic immunoassays.

A variety of antisera are available for analyzing imunoreactivity of theproduct, including but not limited to L4 monoclonal antibody, A5monoclonal antibody, polyclonal antisera raised against purified fusionprotein or G protein as described in Section 6 or 9, infra.

The protein should be immunoreactive whether it results from theexpression of the entire gene sequence, a portion of the gene sequenceor from two or more gene sequences which are ligated to direct theproduction of chimeric proteins. This reactivity may be demonstrated bystandard immunological techniques, such as radioimmunoprecipitation,radioimmune competition, or immunoblots.

Once the RS virus fusion or G protein related protein is identified, itmay be isolated and purified by standard methods includingchromatography (e.g., ion exchange, affinity, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

5.6. Determination of the Immunopotency of the Recombinant Product

Immunopotency of the RS virus fusion or G protein related product can bedetermined by monitoring the immune response of test animals followingimmunization with the purified protein, synthetic peptide or protein. Incases where the RS virus fusion protein or G protein related protein isexpressed by an infectious recombinant virus, the recombinant virusitself can be used to immunize test animals. Test animals may includebut are not limited to mice, rats, rabbits, primates, and eventuallyhuman subjects. Methods of introduction of the immunogen may includeoral, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal or any other standard routes of immunizations.The immune response of the test subjects can be analysed by threeapproaches: (a) the reactivity of the resultant immune serum toauthentic RS viral antigens, as assayed by known techniques, e.g.,enzyme linked immunosorbant assay (ELISA), immunoblots,radioimmunoprecipitations, etc., (b) the ability of the immune serum toneutralize RS virus infectivity in vitro (see Section 6, infra), (c) theability of the immune serum to inhibit virus fusion in vitro (seeSection 6, infra) and (d) protection from RS virus infection (seeSection 6, infra).

5.7. Formulation of a Vaccine

Many methods may be used to administer the vaccine formulationsdescribed herein to an animal or a human. These include, but are notlimited to: oral, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous and intranasal routes. The secretory IgAantibodies produced by the mucosal associated lymphoid issue may play amajor role in protection against RS virus infection by preventing theinitial interaction of the pathogens with the mucosal surface, or byneutralizing the important epitopes of the pathogens that are involvedin infection/or spreading of the disease. Stimulation of mucosal immuneresponses, including production of secretory IgA antibodies may be ofmajor importance in conferring protection against lower and upperrespiratory tract infection. When a live recombinant virus vaccineformulation is used, it may be administered via the natural route ofinfection of the parent wild-type virus which was used to make therecombinant virus in the vaccine formulation.

5.7.1. Subunit Vaccine Formulations

The proteins and polypeptides of the present invention related to aneutralizing and/or fusion epitope of the fusion protein of RS virus areuseful as immunogens in a subunit vaccine to protect against lowerrespiratory disease and other disease symptoms of RS virus infection.Subunit vaccines comprise solely the relevant immunogenic materialnecessary to immunize a host. Vaccines prepared from geneticallyengineered immunogens, chemically synthesized immunogens and/orimmunogens comprising authentic substantially pure RS virus fusionprotein or fragments thereof alone or in combination with similarlyprepared RS virus G protein or fragments thereof, which are capable ofeliciting a protective immune response are particularly advantageousbecause there is no risk of infection of the recipients.

Thus, the RS virus fusion protein and/or G protein related proteins andpolypeptides can be purified from recombinants that express theneutralizing and/or fusion epitopes. Such recombinants include any ofthe previously described bacterial transformants, yeast transformants,cultured cells infected with recombinant viruses or cultured mammaliancells such as Chinese hamster ovary cells that express the RS virusfusion protein epitopes (see Section 5.3, supra). Additionally therecombinants include recombinant attenuated enterovasive bacteriacontaining a DNA sequence which encodes a neutralizing and/or fusionepitope of Respiratory Syncytial Virus fusion protein or a neutralizingepitope of Respiratory Syncytial Virus G protein. Such recombinants areprepared using methods similar to those described in U.S. patentapplication Ser. No. 104,735. These recombinant attenuatedenteroinvasive bacteria are particularly suited for oral vaccineformulations. The recombinant protein or poypeptides can comprisemultiple copies of the epitope of interest.

Alternatively, the RS virus fusion protein and/or G protein relatedprotein or polypeptide can be chemically synthesized (see Section 5.2,supra). In yet another alternative embodiment, the RS virus fusionprotein related protein or polypeptide or G related protein can beisolated in substantially pure form from RS virus or cultures of cellsinfected with RS virus (see, for example, Section 6.1, or Section 10,infra).

Regardless of the method of production, the RS virus fusion protein or Gprotein, related protein or polypeptide is adjusted to an appropriateconcentration and can be formulated with any suitable vaccine adjuvant.The polypeptides and proteins may generally be formulated atconcentrations in the range of 0.1 ug to 100 ug per kg/host.Physiologically acceptable media may be used as carriers. These include,but are not limited to: sterile water, saline, phosphate buffered salineand the like. Suitable adjuvants include, but are not limited to:surface active substances, e.g., hexadecylamine, octadecylamine,octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammoniumbromide, N,N-dioctadecyl-N,-N-bis(2-hydroxyethyl-propane diamine),methoxyhexadecyglycerol, and pluronic polyols; polyamines, e.g., pyran,dextransulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g.,muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineralgels, e.g., aluminum hydroxide, aluminum phosphate, etc. The immunogenmay also be incorporated into liposomes or conjugated to polysaccharidesand/or other polymers for use in a vaccine formulation.

In yet another embodiment of this mode of the invention, the RS virusfusion protein related protein or polypeptide is a hapten, i.e., amolecule which is antigenic in that it reacts specifically orselectively with cognate antibodies, but is not immunogenic in that itcannot elicit an immune response. In such case, the hapten may becovalently bound to a carrier or immunogenic molecule; for example, alarge protein such as protein serum albumin will confer immunogenicityto the hapten coupled to it. The hapten-carrier may be formulated foruse as a subunit vaccine.

The polypeptides and proteins of the present invention may be used whenlinked to a soluble macromolecular carrier. Preferably, the carrier andthe polypeptides and proteins of the present invention are in excess offive thousand daltons after linking. More preferably, the carrier is inexcess of five kilodaltons. Preferably, the carrier is a polyamino acid,either natural or synthetic, which is immunogenic in animals, includinghumans. The manner of linking is conventional. Many linking techniquesare disclosed in U.S. Pat. No. 4,629,783 which is incorporated herein byreference. Many cross-linking agents are disclosed in 1986-87 HandbookAnd General Catalog, Pierce Chemical Company, (Rockford, Ill.) pages 311to 340.

In yet another embodiment of this mode of the invention the immunogen ofthe vaccine formulation comprises a mixture of polypeptides and proteinsrelated to the RS virus fusion protein and the G protein of one or morevirus subtypes.

5.7.2. Viral Vaccine Formulations

Another purpose of the present invention is to provide either a liverecombinant viral vaccine or an inactivated recombinant viral vaccinewhich is used to protect against lower respiratory infections and otherdisease symptoms of RS virus. To this end, recombinant viruses areprepared that express RS virus fusion protein related epitopes (seeSection 5.2, supra). Where the recombinant virus is infectious to thehost to be immunized but does not cause disease, a live vaccine ispreferred because multiplication in the host leads to a prolongedstimulus, therefore, conferring substantially long-lasting immunity. Theinfectious recombinant virus when introduced into a host can express theRS virus fusion protein related protein or polypeptide fragment from itschimeric gene and thereby elicit an immune response against RS virusantigens. In cases where such an immune response is protective againstsubsequent RS virus infection, the live recombinant virus, itself, maybe used in a preventative vaccine against RS virus infection. Productionof such recombinant virus may involve both in vitro (e.g., tissueculture cells) and in vivo (e.g.. natural host animal) systems. Forinstance, conventional methods for preparation and formulation ofsmallpox vaccine may be adapted for the formulation of live recombinantvirus vaccine expressing an RS virus fusion protein related protein orpolypeptide.

Multivalent live virus vaccines can be prepared from a single or a fewinfectious recombinant viruses that express epitopes of organisms thatcause disease in addition to the epitopes of RS virus fusion protein.For example, a vaccinia virus can be engineered to contain codingsequences for other epitopes in addition to those of RS virus fusionprotein. Such a recombinant virus itself can be used as the immunogen ina multivalent vaccine. Alternatively, a mixture of vaccinia or otherviruses, each expressing a different gene encoding for an epitope of RSvirus fusion protein and an epitope of another disease causing organismcan be formulated in a multivalent vaccine.

Whether or not the recombinant virus is infectious to the host to beimmunized, an inactivated virus vaccine formulation may be prepared.Inactivated vaccines are "dead" in the sense that their infectivity hasbeen destroyed, usually by chemical treatment (e.g., formaldehyde).Ideally, the infectivity of the virus is destroyed without affecting theproteins which are related to immunogenicity of the virus. In order toprepare inactivated vaccines, large quantities of the recombinant virusexpressing the RS virus fusion protein related protein or polypeptidemust be grown in culture to provide the necessary quantity of relevantantigens. A mixture of inactivated viruses which express differentepitopes may be used for the formulation of "multivalent" vaccines. Incertain instances, these "multivalent" inactivated vaccines may bepreferable to live vaccine formulation because of potential difficultieswith mutual interference of live viruses administered together. Ineither case, the inactivated recombinant virus or mixture of virusesshould be formulated in a suitable adjuvant in order to enhance theimmunological response to the antigens. Suitable adjuvants include, butare not limited to: surface active substances, e.g., hexadecylamine,octadecyl amino acid esters, octadecylamine, lysolecithin,dimethyl-dioctadecylammonium bromide, N,N-dicoctadecyl-N'-N-bis(2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronicpolyols; polyamines, e.g., pyran, dextransulfate, poly IC, polyacrylicacid, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine,tuftsin; oil emulsions; and mineral gels, e.g., aluminum hydroxide,aluminum phosphate, etc.

5.7.3. Passive Immunity and Anti-Idiotypic Antibodies

Instead of actively immunizing with viral or subunit vaccines, it ispossible to confer short-term protection to a host by the administrationof pre-formed antibody against an epitope of RS virus fusion protein oran epitope of the RS virus G protein. Thus, the vaccine formulations canbe used to produce antibodies for use in passive immunotherapy. Humanimmunoglobulin is preferred in human medicine because a heterologousimmunoglobulin may provoke an immune response to its foreign immunogeniccomponents. Such passive immunization could be used on an emergencybasis for immediate protection of unimmunized individuals exposed tospecial risks, e.g., young children exposed to contact with RS viruspatients. Alternatively, these antibodies can be used in the productionof anti-idiotypic antibody, which is turn can be used as an antigen tostimulate an immune response against RS virus fusion protein or Gprotein epitopes.

5.8. Diagnostic Assays

Yet another purpose of the present invention is to provide reagents foruse in diagnostic assays for the detection of antigens of the RS virusfusion protein or G protein (and hence RS virus) or for the detection ofantibodies to the RS virus fusion protein or G protein in various bodyfluids of individuals suspected of RS virus infection.

5.8.1. Immunoassays

In one mode of this embodiment, the RS virus fusion protein or G proteinrelated proteins, polypeptides and peptides of the present invention maybe used as antigens in immunoassays for the detection of RS virus invarious patient tissues and body fluids including, but not limited to:blood, spinal fluid, sputum, nasal secretions, secretions of therespiratory tract, etc.

The proteins, polypeptides and peptides of the present invention may beused in any immunoassay system known in the art including, but notlimited to: radioimmunoassays, ELISA assay, "sandwich" assays,precipitin reactions, gel diffusion immunodiffusion assays,agglutination assays, fluoresent immunoassays, protein A immunoassaysand immunoelectrophoresis assays, to name but a few. U.S. Pat. No.4,629,783 and patents cited therein also describe suitable assays.

5.8.2. Nucleic Acid Hybridization Assay

In another mode of this embodiment, the novel nucleotide sequence of thegene or gene fragment encoding the RS virus fusion protein relatedproteins polypeptides and peptides of the present invention may be usedas probes in nucleic acid hybridization assays for the detection of RSvirus in various patient body fluids, including but not limited to:blood, sputum, nasal secretions, secretions of the respiratory tract,etc.

The nucleotide sequences of the present invention may be used in anynucleic acid hybridization assay system known in the art including, butnot limited to: Southern blots (Southern, 1975, J. Mol. Biol. 98:508);Northern blots (Thomas et al., 1980, Proc. Nat'l Acad. Sci. USA77:5201-05); colony blots (Grunstein et al., 1975, Proc. Nat'l Acad.Sci. USA 72:3961-65), etc.

The following Examples are offered for the purpose of illustrating thepresent invention and are not to be construed to limit the scope of thepresent invention.

6. Protection of Animals: RS Virus Fusion Protein 6.1. GeneralProcedures 6.1.1. Isolation of Fusion Protein

Substantially pure RS virus fusion protein suitable for use as animmunogen in a subunit vaccine formulation was prepared essentiallyaccording to the procedure of Walsh et al. (1985, J. Gen. Virol. 66:409-15). The procedure may be summarized as follows: Cells were infectedwith an RSV strain. The cells were lysed in a non-denaturing detergentbuffer containing 1% Triton X-100 and deoxycholate. The cell lysateswere clarified by centrifugation. Protein was purified from the celllysates by immunoaffinity purification. A monoclonal antibody againstthe protein was coupled to beads and a column was constructed with thosebeads. RSV-infected cell lysates were applied to the column, and thecolumn was washed with PBS containing 0.1% Triton X-100. Protein boundto the column was eluted with 0.1M glycine, pH 2.5, 0.1% Triton X-100.Elution samples were buffered with Tris and analyzed for the presence ofprotein. Fractions containing the protein were pooled and dialyzedagainst PBS. Rabbit polyclonal antiserum (previously obtained fromrabbits immunized with the RSV fusion protein) and a monoclonalantibody, both specific for the RSV fusion protein, were used to verifythe identity of the purified dimeric RSV fusion protein by Westernblotting. Purified protein has been prepared from three different virusstrains including Long, A2 and 18537 and from two different cell lines,HEp-2 (ATCC No. CCL23) and Vero (ATCC No. CCL81). The protein derivedfrom all sources is highly immunogenic and when used as an immunogenproduces antibody which is virus neutralizing and antifusing. Theelectrophoretic behavior of the purified protein derived from the Longstrain of virus in HEp-2 cells can be seen in FIG. 2 under a variety ofdenaturing conditions. The protein is substantially pure ofcontaminating viral or cellular proteins.

The fusion glycoprotein purified as described above was found to havelipid covalently associated with the F₁ subunit. This was demonstratedby infecting Vero cells with the A2 strain of RS virus and labeling thecultures with [9,10-³ H]-palmitic acid. The purified protein asdemonstrated to have ³ H-palmitic acid associated with the 140,000dalton dimeric form of the protein based on PAGE and autoradiography.Furthermore, treatment of the fusion protein with heat alone or withheat plus reduction of disulfide bonds showed that the ³ H-palmitic acidwas associated with the 70,000 and 48,000 dalton (F₁ subunit) forms ofthe protein.

When ³ H-palmitic acid labeled protein was extracted withchloroform:methanol (2:1 v/v), nearly all of the label remainedassociated with the protein and was not extracted as free lipid into thechloroform:methanol. Therefore, the palmitic acid is covalentlyattached. When the protein was first treated with 1 M hydoxylamine at pH7.0, the palmitic acid was then extracted into chloroform:methanol. Thusthe protein-lipid bond is broken by hydroxylamine showing a covalentester linkage. Since the bond was broken at neutral pH, a thioesterlinkage through cysteine on the protein is the most likely bond althoughother ester bonds could be formed.

In addition, preliminary experiments suggest that myristic acid is alsopresent in the RS virus F Protein. Purified F protein was hydrolysed in1M methanolic HCl at 80° C. for 24 hours; released lipid was extractedin hexane and analyzed by gas chromatography (GC) (Perkin Elmer 8500)using a fused silica column bonded with methyl silicone. The GC spectraobtained indicated the presence of mysristic acid on the purified Fprotein.

The carbohydrate nature of the purified fusion protein obtained asdescribed above was also characterized. The purified protein derivedfrom the Long strain of RS virus obtained from infected HEp-2 cells wasanalyzed as follows: The purified protein was methanolyzed withHCl-containing methanol, fully acetylated, O-deacylated, and finallyper-O-(trimethylsilyl)ated. The sugar residues were identified andquantitated by gas liquid chromatorgraphy (Reinhold, 1972, Methods inEnzymology, 25:244-49). The protein was found to have 5.75% totalcarbohydrate by weight. The sugar composition by percentage of totalsugar was: 11.5% fucose, 3.3% xylose, 26.2% mannose, 9.8% galactose,9.8% glucose, and 39.3% N-acetyl-glucosamine.

6.1.2. Assays 6.1.2.1. Virus Neutralization Assay

Virus neutralization assays were performed as follows:

Test serum samples and the positive control serum were heat inactivatedat 56° C. for 30 min. Test samples were serially diluted. All sera werethen diluted with an equal volume containing about 50 plague formingunits (PFU) of RS virus, and incubated at 37° C. for one hour. A pool ofadult sera which had previously been characterized by enzymeimmunoassay, neutralization and antifusion assays was used for positivecontrol. Sera which had previously been characterized and was known tobe non-immune was used as negative control.

Each incubated serum-virus mixture was inoculated to HEp-2 cells (ATCCNo. CCL23) in a separate well of 24 well plates and virus adsorption wasallowed to take place for 2 hours at 37° C. The inocula were removed.The cell monolayers were washed and overlayed with modified Eagle'smedium plus 5% fetal bovine serum and 1% Sephadex®, and incubated at 37°C. for 3 days. The overlay medium was removed and the cells were washedwith phosphate buffered saline (PBS).

One ml of chilled PBS-methanol (1:5) solution was added to each well,and the cells were fixed for 30 min. at room temperature. ThePBS-methanol fixative was removed, and one ml per well of 5% Carnation™instant milk in PBS, pH 6.8 (BLOTTO) was added. The plate was incubatedfor 30 minutes at 37° C.

The BLOTTO was removed. One-half ml per well of monoclonal antibodiesagainst RS virus (previously titered and diluted with BLOTTO to aworking concentration) was added, and the plate was incubated at 37° C.for 1 hour. The antibodies were removed, and the fixed cells were washedtwice with BLOTTO, 30 minutes each time.

One-half ml/well of horseradish peroxidase conjugated goat anti-mouseIgG (diluted 1:250 in BLOTTO) was added and the plate was incubated for1 hour at 37° C. The goat antibodies were removed, and the fixed cellswere again washed twice with BLOTTO, 30 minutes each time.

One-half ml/well of a peroxidase substrate solution (0.05% 4chloro-1-napthol, 0.09% H₂ O₂ in PBS pH, 6.8) was added, and color wasallowed to develop for 15-30 minutes at room temperature. The substratesolution was removed, and the wells were washed with water and airdried. The number of plaques in each well was determined.

The neutralization ability of a test serum sample is expressed as thedilution which results in a 60% reduction in plaque formation whencompared to non-immune control serum expressed per ml of serum.

6.1.2.2. Anti-Fusion Assay

Anti-fusion assays were performed as follows:

HEp-2 cells in 48 well plates were infected with RS virus at 25 PFU/wellfor 6 hours at 37° C. After infection, the culture medium was replacedwith fresh culture medium containing 0.1% K6 monoclonal antibodies(sterile filtered ascites fluid) and either a heat-inactivated testserum sample or a heat-inactivated non-immune control serum. K6monoclonal antibody is specific for G glycoprotein of RS virus. Thepresence of K6 in the culture medium prevents virus spread via theculture medium. Sephadex® was added to a final concentration of 1% andthe plate incubated for 3 days at 37° C. The culture medium was removedand the cells were washed with PBS.

One ml of chilled PBS-methanol (1:5) solution was added to each well,and the cells were fixed for 30 min. at room temperature. The fixativewas removed, and one ml per well of BLOTTO was added. The plate wasincubated for 30 minutes at 37° C.

The BLOTTO was removed. One-half ml/well of monoclonal antibodiesagainst RS virus (previously titered and diluted with BLOTTO to aworking concentration) was added, and the plate was incubated for 1 hr.at 37° C. The antibody solution was removed and the fixed cells werewashed twice with BLOTTO, 30 minutes each time.

One-half ml per well of horseradish peroxidase conjugated goatanti-mouse IgG diluted 1:250 in BLOTTO was added, and the plate wasincubated for 1 hour at 37° C. The goat antibodies were removed, and thefixed cells were again washed twice with BLOTTO, 30 minutes each time.

One-half ml per well of a peroxidase substrate solution (PBS pH 6.8containing 0.05% 4 chloro-1-napthol, 0.09% of H₂ O₂) was added, andcolor was allowed to develop for 15-30 minutes at room temperature. Thesubstrate solution was removed, and the wells were washed with water andair dried.

The number and typical size of plaques in the well corresponding to thenon-immune control serum sample were determined. The number of plaquesof a similar size was then determined for the wells corresponding to thetest serum samples. The anti-fusion titer of a test serum sample isexpressed as the dilution which yields a 60% reduction in plaques scoredwhen compared to non-immune control serum expressed per ml of serum.

6.1.2.3. Enzyme Immunoassay (EIA)

Antibody titer in serum samples was determined using an EnzymeImmunoassay (EIA) performed as follows:

RS virus fusion protein was diluted to 200 ng/ml incarbonate-bicarbonate buffer, pH 9.6. One hundred ul of the dilutedantigen was added to each well of rows B-G of a flat-bottomed, 96 wellNunc™ assay plate. In rows A and H, 100 ul of carbonate-bicarbonatebuffer alone was added to each well. The plate was covered and incubatedfor 2 hours at 37° C. with shaking and then stored overnight at 4° C. toimmobilize the antigen.

The supernatants were removed from the Nunc™ assay plate and the platewas washed with 0.1% Tween/PBS pH 7.4 and pat dried.

Three antibody samples were assayed on each plate. Each sample was firstdiluted to a primary dilution in 0.2% Tween, 0.01 M EDTA/PBS pH 7.5(0.2% TWN). The primary dilutions were further serially diluted asfollows in a 96 well U-bottomed Falcon™ plate:

(a) The primary dilutions of the samples were inoculated into row 2 at200 ul/well. Sample 1 was inoculated in triplicate, =.g., in wells A2,B2, and C2; Sample 2 in duplicate e.g., in wells D2, E2; Sample 3 intriplicate e.g. in wells F2, G2, and H2.

(b) 100 ul of 0.2% TWN were inoculated into each well of rows 3-12.

(c) Serial dilutions were created by transferring sequentially 100 ulfrom a well in row 2 to the corresponding well in row 3 (e.g. B2 to B3;C2 to C3), a well in row 3 to the corresponding well in row 4, until row12 was reached.

(d) To row 1, 100 ul of 0.2% TWN was added to each well as control.

One hundred ul of the primary dilutions were transferred from each wellof the Falcon™ plate to the corresponding well in the Nunc™ plate, e.g.,A2 (Falcon™) to A2 (Nunc™). The Nunc™ assay plate was covered andincubated for 1 hour at 37° C. with shaking. The supernatants wereremoved from the assay plate, and the plate was washed with 0.1%Tween/PBS and pat dried.

Goat anti-Mouse IgG alkaline phosphatase conjugate (TAGO™) was dilutedwith 0.3% Tween/PBS pH 7.0 (0.3% TWN) to a working dilution, e.g.,1:1500. The diluted conjugate (100 ul) was added to each well in rows2-12. To row 1, 100 ul of 0.3% TWN were added to each well as control.The plate was covered and incubated for 1 hour at 37° C. with shaking.The inocula was then removed, and the plate was washed with 0.1%Tween/PBS pH 7.4 and pat dried.

To each and every well, 100 ul substrate solution, 1 mg/ml indiethanolamine buffer pH 9.8 (SIGMA-104™) were added. The enzymaticreaction was allowed to take place at room temperature for 1 hour. Thereaction was then stopped by adding 100 ul of 3N NaOH to each well. Theextent of enzymatic reaction was determined by reading the opticaldensity at 410 nm.

Rows A and H served as negative controls because no antigen was present;row 1 was also served as a negative control because no antibodies werepresent.

6.2. Protection of Animals: Homologous and Heterologous Protection

In one experiment, eighteen cotton rats were divided into 6 groups of 3animals each. At week 0, 2 and 4-5, animals were actively immunized byintramuscular injection of I0 ug RS virus fusion protein (140,000daltons), except Group 6 which received only 5 ug protein, in differentadjuvants: Group 1, PBS; Group 2, IFA; Group 3, ISCOM; and Group 4,alum. Group 5 received intramuscular injections of PBS alone and servedas the control group. Group 6 received intramuscular injections of RSvirus fusion protein previously reduced using beta-mercaptoethanol, butwithout any adjuvant.

Serology assays were performed as described in Section 6.1.2, supra onserum samples obtained at week 6-7. Results are presented in Table 1.

Animals were challenged between weeks 6-7 with 1×10⁴ PFU RS virus. Lungswere harvested on day 4 post-challenge. The presence and/or quantity ofRS virus in lung tissues was determined. At day 4 post-infection, thelung and nasal turbinates were removed from the animals and homogenizedin 1-2 ml of virus transport media (MEM, 5% FBS, 2 mM glutamine, 20 mMHEPES, 10% SPG). After centrifugation, the supernatants were seriallydiluted and applied to HEp-2 cells in 24 well tissue culture plates andthe virus grown. Plaques were identified as described in Section6.1.2.1. (Virus Neutralization Assay) and expressed per gram of tissue.Results are also presented in Table 1.

                  TABLE 1                                                         ______________________________________                                        IMMUNOGENICITY OF RSV FUSION                                                  PROTEIN IN COTTON RATS                                                                Serology.sup.b                                                                    Virus           Protection.sup.c                                  Group No.         Neutral-  Anti- Virus                                       (Adjuvant).sup.a                                                                        EIA     ization   fusion                                                                              Present                                                                              Titer                                ______________________________________                                        Group 1   6.2     4.4       2.0   0      0                                    (PBS)                                                                         Group 2   7.3     5.1       2.9   0      0                                    (IFA)                                                                         Group 3   7.4     4.5       2.6   0      0                                    (ISCOM)                                                                       Group 4   7.1     5.2       2.7   0      0                                    (ALUM)                                                                        Group 5   <1.0    <2.0      <1.0  3      4.8                                  (control)                                                                     Group 6   4.9     2.3       2.4   3      1.1                                  (none)                                                                        ______________________________________                                         .sup.a Group 6 received fusion protein treated with beta mercaptoethanol      and no adjuvant.                                                              .sup.b Assays were performed on samples from week 7. Results represent th     geometric mean titer three animals expressed in log.sub.10 units. See tex     for experimental details.                                                     .sup.c Three animals in each of Groups 1-5 and 4 animals in Group 6 were      challenged at weeks 6-7 and lungs were harvested on day 4 postchallenge.      Virus was isolated from the lung tissues. "Virus present" represents the      number of animals in each group in which virus was detectable. "Titer"        represents the geometric mean titer (PFU/gm of tissue) of virus isolated      from lung tissue.                                                        

As demonstrated in Table 1, immunization with RS virus fusion proteinelicited production of antibodies effective virus neutralization and inpreventing fusion. Moreover, results illustrated in Table 1 clearlydemonstrate that lung tissues of immunized animals are effectivelyprotected against subsequent RS virus infection.

When the fusion protein is treated (reduced) with betamercaptoethanol,the subunits are dissociated yielding free F₁ and F₂. When cotton ratswere immunized with 5 ug of the reduced protein without adjuvant, theyalso produced antibody that was neutralizing and had antifusion activity(see Table 1, Group 6), although the level of these activities wasreduced. Similarly, the lungs of these animals were substantiallyprotected from infection, however, with less efficacy. Thus, thesubunits of the fusion glycoprotein, presented in a dissociated form tothe immune system, are able to produce protective immunity. It shouldalso be noted that under these conditions, the F₁ subunit, whichcontains the hydrophobic membrane anchor region, will aggregate forminghigher molecular weight multimers which may enhance immunity.

Human RS virus is subdivided into two subtypes, A and B. The fusionproteins from both subtypes share antigenic determinants and thesedeterminants are highly conserved among RS virus strains. Hence a seriesof experiments were conducted to determine whether immunization with Fprotein from one subtype of human RS virus would confer protectionagainst infection by both subtypes of human RS virus.

In this series of experiments, 30 cotton rats were divided into 4 groupsof experimental animals and 4 groups of control animals. At week 0, 2and 4, experimental animals were actively immunized by intramuscularinjection of 10 ug human RS virus fusion protein (140,000 daltons)obtained from RS virus A2 strain, a subtype A virus. Control animalswere similarly immunized with a placebo immunogen, i.e. PBS.

Serology assays were performed as described in Section 6.1.2, supra, totest the virus neutralization and anti-fusion capabilities of theinduced antibodies against both A and B subtypes of human RS virus.Immunization with subtype A RS virus fusion protein induced antibodieswhich exhibited similiar neutralization and anti-fusion activitiesagainst both subtypes A and B of RS virus (data not shown).

All experimental and control animals were challenged intranasally atweek 6 with human RS virus as follows: Group 1 received strain A2(subtype A) 6.2 log 10 PFU; Group 2, Long strain (subtype A) 6.1 log 10PFU; Group 3, strain 9320 (subtype B) 3.5 log 10 PFU; and Group 4,strain 18537 (subtype B) 5.0 log 10 PFU. At 4 days post-challenge, thelungs were harvested and the virus titer was determined as describedabove. Results are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        PROTECTIVE EFFICACY OF FUSION PROTEIN                                         VACCINE AGAINST VARIOUS SUBTYPES OF RSV                                              Challenge Virus                                                                          Lung Titer (GMT).sup.b                                      Group No..sup.a                                                                        (Subtype)    Experimental.sup.c                                                                         Control.sup.d                              ______________________________________                                        1        A2       (A)     1.1        5.0                                      2        Long     (A)     1.1        4.0*                                     3        9320     (B)     1.43       5.1*                                     4        18537    (B)     1.1        3.5                                      ______________________________________                                         .sup.a Experimental animals in each group were immunized at week 0, 2 and     4 with 10 ug of F protein purified from RS virus strain A2, subtype A.        Control animals in each group similarly received PBS as immunogen. All        animals were challenged at week 6 with RS virus. See text for experimenta     details.                                                                      .sup.b "Lung Titer (GMT)" represents the geometric mean titer (PFU/gm of      tissue) of virus isolated from lung tissue.                                   .sup.c N = 5.                                                                 .sup.d N = 3, except * which designated N = 2.                           

As demonstrated in Table 2, immunization with fusion protein from asubtype A virus strain, induced significant protection against both ahomologous subtype A virus and a heterologous sybtype B virus in allcases. Thus it is clear that immunization with F protein from onesubtype RS virus confers protection against both subtypes of human RSvirus.

6.3. Protection of Baboons

Twelve juvenile baboons were divided into 3 groups of 4 animals each.Animals were injected intramuscularly with 20 ug purified RS virusfusion protein at one month intervals for 3 months as follows: Group 1received immunogen in PBS; Group 2, immunogen in alum; and Group 3, PBSalone (control group).

Serology assays were performed two weeks after the third immunization asdescribed in Section 6.1.2, supra. Results are presented in Table 3.

At 2 weeks post-immunization, 3-4 animals/group were challenged with5.32 PFU of Strain A2 RS virus by direct innoculation into the lungs.Lung lavages were assayed at day 2 post-infection. Lung lavages wereobtained by direct transfer of media into the lungs and collection bytube aspiration. The virus was titered as described in Section 6.2,supra. Results are also presented in Table 3.

                  TABLE 3                                                         ______________________________________                                        IMMUNOGENICITY OF RSV FUSION PROTEIN IN                                       BABOONS                                                                       Group No.                                                                             Serology.sup.a                                                        Immunogen       Virus       Anti  Virus Assay.sup.b                           (Adjuvant)                                                                            EIA     Neutralization                                                                            fusion                                                                              +   -   TITER                               ______________________________________                                        Group 1 5.01    2.5         1.83  3   1   1.05                                (PBS)                                                                         Group 2 6.0     3.34        2.56  0   4   0.00                                (ALUM)                                                                        Group 3 <1.0    <2.0        <1.0  3   0   2.14                                (control)                                                                     ______________________________________                                         .sup.a Assays were performed two weeks after the last (third)                 immunization. Results represent the geometric mean titer 4 animals            expressed in log.sub.10 units. (See text for experimental details).           .sup.b Strain A2 RS virus (5.32 PFU) was inoculated directly into the         lungs of 3-4 immunized animals in each group at two weeks post                immunization. Lung lavages were assayed at day 2 postinfection. "+"           represents the number of animals with detectable RS virus in lung lavages     "-" represents the number of animals without detectable RS virus in lung      lavages. "Titer" is expressed as the geometric mean titer (PFU/gm) of         tissue of virus isolated from lun g tissue.                              

As shown in Table 3, immunization of juvenile baboons with purified RSvirus fushion protein elicited antibodies which were able to neutralizevirus and prevent fusion. As further clearly demonstrated, immunizedanimals were protected against subsequent RS virus infection when alumwas used as an adjuvant (Group 2).

In another series of experiments, juvenile baboons were divided into anumber of groups and immunized by intramuscular injection of purified RSvirus fusion protein as follows: 4 animals received 5 ug of immunogen inalum and 11 animals received 20 ug of immunogen in alum on days 0, 28and 56; 6 animals received 100 ug of immunogen in alum on day 0, 20 ugon day 28 and 20 ug on day 56. Twelve animals received PBS alone on day0, 28 and 56 and served as controls. All animals were challenged with<10⁶ PFU of strain A2 RS virus on day 70 by direct inoculation into thelungs. Lung lavages were collected as described above on days 2, 3 and 4post-challenge Results from this series of experiments are summarized inTable 4.

                  TABLE 4                                                         ______________________________________                                        SUMMARY OF BABOON PROTECTION                                                             RSV Isolation From Lung Lavages.sup.a                              Immunogen    N      (+)        (-)  Titer                                     ______________________________________                                        F (alum)     21      2         19   <0.7                                      PBS          12     12          0    3.0                                      ______________________________________                                         .sup.a "(+)" represents the number of baboons in which RS virus was           detected in lung lavage. "(-)" represents the number of baboons in which      no RS virus was detected in lung lavage. "Titer" is expressed as the          geometric mean titer (PFU/ml of tissue) of virus isolated from lung           lavage.                                                                  

As summarized in Table 4, baboons were efficiently protected against RSvirus infection by immunization with RS virus fusion protein. During thecourse of immunization and subsequent challenge experiments, bloodchemistries and hematologic assays showed no sign of adverse reactionsthroughout the course of the experiments.

6.4. Protection Against Bovine RS Virus

A crude preparation of human RS virus fusion protein (herein termed"crude human RS virus F protein" was prepared as follows: Vero cellsinfected with RS virus A2 strain were extracted with a solutioncontaining 50 mM Tris; 0.15M NaCl; 1% Triton X-100 and 2% deoxycholate;pH 7.4 (Lysis Buffer). The F protein was obtained from the spent cellculture medium by solubilization of PEG pelleted virus using the LysisBuffer. The resulting crude preparation was clarified by centrifugation.An immunoaffinity purified preparation was prepared as described inSection 6.1.1.

Fifteen cows were divided into 5 groups of 3 animals each. Animals wereinjected intramuscularly with human RS virus fusion protein on days 0and 21 as follows: Group 1 received crude human RS virus F protein (5ug); Group 2, crude human RS virus F protein (20 ug); Group 3, purifiedhuman RS virus F protein, (20 ug); Group 4, a USDA approved bovine RSvirus vaccine commercially available from Diamond Scientific Co. (DesMoines, Iowa) (2 ml on days 0 and 21) and Group 5, PBS alone (control).

EIA's were performed on serum samples obtained on days 0, 10, 21 and 33post-immunization as described in Section 6.1.2., supra, F protein fromRS virus strain A2 as antigen. Results are presented in Table 5.

                  TABLE 5                                                         ______________________________________                                        IMMUNOGENICITY OF RS VIRUS FUSION PROTEIN IN                                  COWS                                                                                       Serology (EIA).sup.b                                                          Day                                                              Immunogen.sup.a                                                                              0        10       21     33                                    ______________________________________                                        HRSV F protein (5 ug)*                                                                       --       --       3.6 (2/3)                                                                            5.8                                   HRSV F Protein (20 ug)*                                                                      --       3.8 (2/3)                                                                              4.2 (3/3)                                                                            6.2                                   HRSV F protein (20 ug)                                                                       --       3.8 (2/3)                                                                              4.6 (2/3)                                                                            5.9                                   Diamond Vaccine (BRSV)                                                                       2.2 (1/3)                                                                              3.0 (1/3)                                                                              3.5 (2/3)                                                                            4.7                                   PBS            --       --       --     --                                    ______________________________________                                         .sup.a Cows were immunized at days 0 and 21 with either crude (*) or          immunoaffinity purified F protein and the immunogenicity was compared wit     that of a commerically available USDA approved BRSV vaccine (Diamond          Scientific Co. vaccine). See text for details.                                .sup.b Results represent the geometric mean titer of 3 animals expressed      in log.sub.10 units. The ratio in the parenthesis is the number of ELISA      positive animals. "--" indicates that the titer was not detectable, i.e.,     less than 1.6.                                                           

The data presented in Table 5 shows that the human RS virus fusionprotein obtained as described in Section 6.1 and after immunoaffinitypurification elicits high levels of antibodies. Comparison with acommercially available bovine RS virus vaccine showed that the human RSvirus protein was as immunogenic or more immunogenic in cows than theBRSV vaccine.

The protective immunogenicity of the human RS virus fusion proteinagainst bovine RS virus was investigated in another series ofexperiments. Twenty five cotton rats were divided into 2 groups of 10animals each and one group of 5 animals each. Animals were immunizedintramuscularly on days 0 and 21 as follows: Group 1 received thecommercially available BRSV vaccine (Diamond); Group 2, human RS virusLong strain F and G protein (10 ug each)/Alum (affinity purified F and Gproteins were obtained as described in Sections 6.1.1 and 10.1,respectively); and Group 3, PBS/Alum (control). Serum samples wereobtained and serological assays were performed as described in Section6.2, supra, except that BRSV (strain 3758) was used for theneutralization and anti-fusion assays. Results are presented in Table 6.

                  TABLE 6                                                         ______________________________________                                        PROTECTIVE EFFICACY OF HUMAN RS                                               FUSION PROTEIN AGAINST BOVINE RS VIRUS.sup.a                                         Serology.sup.b                                                                EIA       Neutralization                                                                            Anti-fusion                                             Day       Day         Day                                              Immunogen                                                                              21      28      21    28    21    28                                 ______________________________________                                        Diamond  4.2     5.3     n.d.  2.6   n.d.  <1:20                                       (10)    (6)           (6)         (6)                                F&G/Alum.sup.c                                                                         6.0     7.0     n.d.  4.3   n.d.  2.2                                         (10)    (8)           (8)         (8)                                PBS/Alum <1:100  <1:100  n.d.  <1:20 n.d.  <1:20                                        (5)    (10)          (4)         (4)                                ______________________________________                                         .sup.a See text for experimental details.                                     .sup.b Results represent the geometric mean of 4 to 10 animals expressed      in log 10 units. The number in parentheses indicates the number of            animals.                                                                      .sup.c The dose was 10 ug of each protein.                               

As demonstrated in Table 6, human RS virus fusion (and G) proteinelicited very high titer of antibodies that showed high neutralizing andanti-fusion activities against BRSV. On the other hand, the Diamond BRSVvaccine elected antibodies that had no detectable anti-fusion activityand significantly lower neutralization activity. Thus, the resultspresented in Table 6 clearly indicate that human RS virus fusion proteinelicits antibodies that neutralize BRSV and prevent fusion induced byBRSV. Hence, the human RS virus fusion protein elicits a protectiveimmune response against BRSV.

6.5. Avoidance of Enhanced Disease

Immunization or vaccination with formalin inactivated RS virus (Lot 100,Pfizer) has been associated with the development of atypical and moresevere disease when immunized subjects have been subsequently infectedwith RS virus. (Kim et al., 1969, Am. J. Epidemol. 89:422-34; Chin etal., 1969, Am. J. Epidemol. 89:449-63). Such potentiation of RS virusinfection has been a major hurdle for the development of an efficaciousand safe RS virus vaccine formulation for human and animal subjects.

Prince et al. (1985, J. Virol. 57:721-28) developed an animal model forthe assessment of the potentiation or enhancement of RS virus induceddisease. The cotton rat animal model developed by Prince was employed toinvestigate whether or not similar potentiation or enhancement ofsubsequent disease was associated with the use of RS virus F protein asan immunogen according to one embodiment of the present invention.Briefly, cotton rats were immunized by intramuscular injection of eitherRS virus F protein, with or without Alum as an adjuvant, or one of avariety of immunogens as indicated in Table 7. All animals receivedthree injections either on week 0, 1 and 2 or on week 0, 2, and 4,except for one group which received only one immunization. One to twoweeks following the last immunization, animals were challenged with 10⁴-10⁶ PFU/animal of RS virus. At a 1 and/or 4 days post-challenge, thelungs of the animals were examined for morphological, inflammatory andpathological changes in alveolar ducts and/or alveoli according to themethod of Prince et al., supra. The overall pathology of large and smallairways of lungs were scored using a scheme ranging from mild (+1),moderate (+2), severe (+3) to very severe (+4). Results are presented inTable 7.

                  TABLE 7                                                         ______________________________________                                        ENHANCED PATHOLOGY COTTON RAT MODEL:                                          FREQUENCY OF OCCLUDED SMALL AIRWAYS                                                               Alveolar and/or                                           Immunogen Sample    Alveoli Pathology.sup.b                                   (Dose).sup.a                                                                            Challenge Day    N    +    (%)  Severity                            ______________________________________                                        Lot-100 (undil)                                                                         RSV       1      2    1    (50) 1.5                                 Lot-100 (undil)                                                                         RSV       4      3    2    (67) 1.5                                 RSV-      RSV       1      18   13   (72) 2.0                                 formalin                                                                      (undil-1:625)***                                                              RSV-      RSV       4      17   11   (65) 1.5                                 formalin                                                                      (undil-1:625)***                                                              RSV-live  RSV       1      13   2    (15) 1.0                                 (10.sup.1 -10.sup.6 PFU)                                                      RSV-live  RSV       4      15   3    (20) 1.0                                 (10.sup.1 -10.sup.6 PFU)                                                      F (Alum)* RSV       4      52   0    0    --                                  (0.2-20 ug)                                                                   F (5 ug)  RSV       1      3    0    0    --                                  F (0.2-10 ug)                                                                           RSV       4      23   0    0    --                                  F** (1-20 ug)                                                                           RSV       4      11   1     (9) 0.5                                 Medium    RSV       1      2    0                                             Medium    RSV       4      2    1    (50) 1.0                                 PBS       RSV       1      4    1    (25) 1.0                                 PBS       RSV       4      22   3    (14) 1.0                                 None      RSV       1      3    0    0    --                                  None      RSV       4      3    0    0    --                                  None*     Medium    4      3    0    0    --                                  None*     None                                                                          None or   4      9    0    0    --                                            PBS                                                                 ______________________________________                                         .sup.a All animals were immunized by a 200 ul intramuscular injection of      immunogen three times at weeks 0, 1, and 2, except for those designated *     which were immunized at weeks 0, 2 and 4 and ** which received a single       intramuscular injection at week 0. "***" indicates that the undiluted         virus was 10.sup.6 -10.sup.7 PFU.                                             .sup.b "Day" indicates the day postchallenge on which the pathology of        lung avelolar ducts and/or alveoli were evaluated. "N" indicates the tota     number of animals evaluated. "+" indicates the number of animals in which     pathological changes were noted, indicating enhanced or potentiated RS        virus induced disease. "(%)" represents the percent of animals in which       enhanced RS virus induced disease was observed. "Severity"represents the      mean histopathological score on a scale of according to the method of         Prince et al., supra 0 to +4)                                            

As demonstrated in Table 7, animals which were immunized with RS virusfusion protein according to one embodiment of the present invention, didnot exhibit pathological changes in lung tissues, including alveolarducts and/or alveoli. On day 4, only 1 out of 86 animals immunized withfusion protein showed less than mild pathological change. On the otherhand, of animals immunized with commercially available RS virus vaccineLot 100 or formalin-inactivated RS virus, greather than 65% of treatedanimals had mild to moderate pathological deterioration and/orinflammation of lung tissues. Additionally, 20% of animals immunizedwith live RS virus showed at least mild pathological deteriorationand/or inflamation of lung tissues. Thus, the use of RS virus fusinprotein according to the present invention avoided the enhancement orpotentiation of disease induced by subsequent RS virus infection. Hencethe vaccine formulations according to the present invention are bothsafe and efficacious.

7. Protection of Humans: RS Virus Fusion Protein

FDA approved human phase I clinical studies were conducted to evaluatethe safety and immunogenicity of RS virus fusion protein vaccine inhuman adult volunteers. The fusion protein from the RS virus strain A2,was purified as described in Section 6.1, supra, compounded with alum,and injected intramuscularly in the following doses: Group 1 received 5ug (N=15), Group 2, 15 ug (N=16); and Group 3, 45 ug (N=9). One monthlater, 6 volunteers from Group 1 and 7 volunteers from Group 2 wereboosted with a repeat injection of the respective amounts of RS virusfusion protein received at the first immunization. The schedule forvaccinations and collection of serum samples from volunteers isillustrated in Table 8.

                  TABLE 8                                                         ______________________________________                                        DESCRIPTIVE DATA FROM PHASE I CLINICAL                                        TRIAL OF RS VIRUS F PROTEIN IN ADULTS                                                       RS Virus F Protein                                              Volunteers                                                                             5 ug       15 ug      45 ug   Total                                  ______________________________________                                        (vax = 1)                                                                              15         16         9       40                                     (vax = 2)                                                                               6          7                 13                                     Bleed/Vax                                                                     timing:                                                                       A(V)  mean   0      days  0    days  0    days                                B     mean   14.1         14.1       14.0                                     C(V)  mean   32.5         28.8       30.3                                     D     mean   61.6         55.0       58.4                                     E     mean   88.7         83.0       86.4                                     F     mean   166.0        161.4      165.1                                    ______________________________________                                    

The safety data including clinical data and symptoms reported byimmunized human volunteers are summarized in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    PHASE-I CLINICAL TRIAL OF RS VIRUS F PROTEIN IN ADULTS:                       TEMPERATURE AND LOCAL REACTIONS AT VACCINATION SITE                           OCCURRING WITHIN THE FIRST 24 HOURS POST-VACCINATION                                     Number of Individuals by Reaction                                             Vaccine:                                                           Type of    Primary    Secondary                                               Reac-      Dosage:                                                            tion Score 5 ug                                                                             15 ug                                                                             45 ug                                                                             All   5 ug                                                                             15 ug                                                                             All                                        __________________________________________________________________________    Temper-                                                                            0 (≦100° F.)                                                          15 15  9   39                                                                              (100%)                                                                            6  7   13                                                                              (100%)                                   ature                                                                              1 (>100° F.)                                                                 0  0   0   0     0  0   0                                          Redness                                                                            0 (≦ none)                                                                   15 14  9   38                                                                              (97%)                                                                             5  7   12                                                                              (92%)                                    (local)                                                                            1 (1 cm)                                                                            0  1   0   1 (3%)                                                                              0  0   0                                               2 (≧2 cm)                                                                    0  0   0   0     1  0   1 (8%)                                     Swelling                                                                           0     15 14  8   37                                                                              (94%)                                                                             6  7   13                                                                              (100%)                                   (local)                                                                            1 (≦1 cm)                                                                    0  0   0   0     0  0   0                                               2 (≧2 cm)                                                                    0  0   1   2 (6%)                                                                              0  0   0                                          Warmth                                                                             0     15 15  8   38                                                                              (97%)                                                                             6  7   13                                                                              (100%)                                   (Local)                                                                            1 (≦1 cm)                                                                    0  0   0   0     0  0   0                                               2 (≧2 cm)                                                                    0  0   1   1 (3%)                                                                              0  0   0                                          Pain 0 (none)                                                                            10 6   3   19                                                                              (49%)                                                                             3  3   6 (46%)                                    (local)                                                                            1 (touch/                                                                           5  6   5   16                                                                              (41%)                                                                             3  3   6 (46%)                                         movement)                                                                     2 (always)                                                                          0  3   1   4 (10%)                                                                             0  1   1 (8%)                                     __________________________________________________________________________

As demonstrated in Table 9, the adverse reactions (local reactions) tothe immunization were very minimal and/or insignificant. Blood was drawnfrom the volunteers at specified time intervals and tested for theantibody titer specific to the RS virus protein as described above.Results are presented in Table 10.

                  TABLE 10                                                        ______________________________________                                        IMMUNOGENICITY OF RS VIRUS F PROTEIN IN                                       ADULTS EIA SEROLOGY*                                                          Vaccina-   Geometric Mean Titer (Range) [N]                                   tion   Bleed   Group 1     Group 2   Group 3                                  ______________________________________                                        Primary                                                                              A       66.4 (15-176)                                                                             58.9 (21-200)                                                                           90.9                                                    [n = 15]    [n = 16]  (27-299)                                                                      [n = 9]                                         B       160.8.sup.a (30-328)                                                                      229.0.sup.a (40-965)                                                                    415.3.sup.a                                             [n = 15]    [n = 16]  (179-700)                                                                     [n = 9]                                         C       141.3.sup.b (33-276)                                                                      199.9.sup.a (46-828)                                                                    386.2.sup.a                                             [n = 15]    [n = 9]   (153-703)                                                                     [n = 9]                                         D       116.2.sup.c (31-217)                                                                      235.2.sup.a (73-644)                                                                    351.1.sup.b                                             [n = 8]     [n = 9]   (121-645)                                                                     [n = 9]                                         E       119.4.sup.c (35-195)                                                                      180.2.sup.a (60-493)                                                                    349.0.sup.b                                             [n = 9]     [n = 9]   (129-705)                                                                     [n = 9]                                         F       106.1 (25-169                                                                             144.5.sup.b (46-330)                                                                    314.8.sup.b                                             [n = 9]     [n = 9]   (111-538)                                                                     [n = 9]                                  Boost  D       183.4.sup.b (98-302)                                                                      217.7.sup.a (105-117)                                             [n = 6]     [n = 7]                                                   E       166.7.sup.b (103-352)                                                                     217.6.sup.a (99-658)                                              [n = 6]     [n = 7]                                                   F       126.1.sup.c (76-234)                                                                      180.2.sup.b (91-691)                                              [n = 6]     [n = 7]                                            ______________________________________                                         *EIA titer is expressed as titer (X1000). Values represent geometric mean     titer, with the range in parentheses and the number of samples in square      brackets.                                                                     .sup.a Comparison to Bleed A GMT by T test showed significant difference      <  0.001.                                                                     .sup.b Comparison to Bleed A GMT by T test showed significant difference      < 0.01.                                                                       .sup.c Comparison to Bleed A GMT by T test showed significant difference      < 0.05.                                                                  

As shown in Table 10, significantly enhanced quantities of antibodiescompared to the pre-existing antibodies were produced in all groups ,following the immunization with either 5 or 15 or 45 ug of RS virus Fprotein . Hence, the RS virus F protein according to one embodiment ofthis invention, is very immunogenic not only in animals but also inhumans.

8. IDENTIFICATION OF NEUTRALIZING AND/OR FUSION EPITOPE[S] OF RS VIRUSFUSION PROTEIN 8.1. General Procedures

The following protocols were used to define an epitope of RS virusfusion protein which elicits both neutralizing and antifusionantibodies.

8.1.1. Protein Immunoblot (Western Blot) Analysis

Fusion protein subunits F₁ and F₂ were subjected to SDS-PAGE (Laemmli,1970, Nature, 227:680-685). The separated protein subunits in the gelwere electrophoretically transferred to a nitrocellulose sheet using atransfer solution containing 12.1 g Tris-HCl, 56.3 g glycine per 5liters. The nitrocellulose sheet was air dried. The air-driednitrocellulose was incubated at 37° C. sequentially with (i) BLOTTO for15 min., (ii) BLOTTO containing the L4 monoclonal antibody for 15 min.,(iii) BLOTTO for I5 min., (iv) BLOTTO containing secondary antiserum for60 min., and (v) BLOTTO for 15 min.

The secondary antiserum bound to the L4 monoclonal antibody, and waseither labeled with horse radish peroxidase or not labeled. If labeledwith peroxidase, antibody binding was detected by color developmentbrought about by enzymatic reaction with 0.05% 4-chloronapthol, 0.09% H₂O₂ in phosphate buffered saline pH 6.8. If unlabeled, antibody bindingwas detected by autoradiography following ¹²⁵ I- protein A binding tothe antibodies.

8.1.2. Coupling of Peptides to Keyhole Lympet Hemocyanin (KLH) andProduction of Rabbit Antisera

A 1 mg/ml KLH solution was prepared as follows: 4 mg of a resuspendedammonium sulfate precipitate of KLH (containing 37 mg of protein/ml) wascentrifuged for 5 minutes at 4° C. in a microfuge. The supernatant wasdiscarded and the pellet redissolved in 3 ml of 0.1M NaHCO₃. Thesolution was dialyzed against 0.1M NaHCO₃ with 2 changes. The volume ofthe dialyzed KLH solution was adjusted to 4 ml to make a 1 mg/ml KLHsolution.

Four ul of a 1 mg/ml solution of a synthetic polypeptide were added tothe KLH solution, and mixed at room temperature for 1 hour. Four ml of25% aqueous glutaraldehyde were added to the mixture, and mixed foranother 24 hours at room temperature. Twenty ul of 25% aqueousglutaraldehyde were again added to the mixture and mixed for a further72 hours at room temperature. The glutaraldehyde cross-linked syntheticpolypeptide--KLH was dialyzed overnight against PBS with several changesof the dialysis buffer.

Rabbits were immunized with 250 ug of protein in complete Freund'sadjuvant, and boosted 2-3 times at 2 week intervals with 250 ug ofprotein in incomplete Freund's adjuvant.

8.1.3. Proteolytic Cleavage of Fusion Protein Trypsin Digestion withoutL4 Protection

Purified fusion protein in 50 mM Tris, pH 7.1, .05% SDS and 0.1%beta-mercaptoethanol was heated for 5 minutes at 100° C. Trypsin wasadded to the cooled, denatured fusion protein sample and incubated at37° C. for 2 hours Enzyme to substrate ratios of 1:1000, 1:2500, and1:5000 were used. Unless otherwise stated, all proteinases are addedfrom a 1 mg/ml stock solution.

Trypsin Digestion with L4 Protection

Equal molar amounts of purified fusion protein and L4 monoclonalantibody were mixed and set on ice for 1 hour. Trypsin was added to themixture in the presence of 50 mM Tris, pH 7.1, and incubated at 37° C.for 2 hours. An enzyme to substrate ratio of 1:10 was used.

Trypsin/Arg-C Digestions with L4 Protection

Equal molar amounts of purified fusion protein and L4 monoclonalantibody were mixed and set on ice for 1 hour. Trypsin digestion at anenzyme to substrate ratio of 1:10 was carried out as described inExample 2.2. The trypsin-digested fusion protein was heated for 5minutes at 100° C., cooled and digested further by Arg-C at enzyme tosubstrate ratios of 1:2, 1:5, 1:10 at 37° C. for 2 hours.

Arg-C/Arg-C Digestions with L4 Protection

Equal molar amounts of purified fusion protein and L4 monoclonalantibody were mixed and set on ice for 1 hour. A first Arg-C digestionof the mixture at an enzyme to substrate ratio of 1:3 was performed inthe presence of 20 mM NH₄ HCO₃ for 2 hours at 37° C. Then the reactionmixture was reduced by adding 2-mercaptoethanol to a final concentrationof 0.1% and heated for 5 minutes at 100° C. After cooling, a secondArg-C digestion at an enzyme to substrate ratio of 1:6 for 2 hours at37° C. was carried out.

Lys-C/Arg-C Digestions with L4 Protection

The same protocol was followed as in Example 2.4 above except that Lys-Cat an enzyme to substrate ratio of 1:2 was used in the first digestion.

8.1.4. Dot Blot Analysis

Dot blot analysis was performed on synthetic polypeptides as follows:

Polypeptides (up to 20 ug) were spotted on the nitrocellulose sheets.The nitrocellulose sheet was air dried. The air-dried nitrocellulose wasincubated at 37° C. sequentially with (i) BLOTTO (5% Carnation™ instantmilk in phosphate buffered saline pH 6.8) for 15 min., (ii) BLOTTOcontaining the L4 monoclonal antibody for 0.25-72 hrs, (iii) BLOTTO for15 min., (iv) BLOTTO containing secondary antiserum for 60-120 min., and(v) BLOTTO for 15 min.

The secondary antiserum bound to the L4 monoclonal antibody, and waseither labeled with horse radish peroxidase or not labeled. If labeledwith peroxidase, antibody binding was detected by color developmentbrought about by enzymatic reaction with 0.06% 4-chloronapthol, 0.3% H₂O₂ in phosphate buffered saline pH 6.8. If unlabeled, antibody bindingwas detected by autoradiography following ¹²⁵ I- protein A binding tothe antibodies.

8.2. Mapping by Defined Proteolytic Cleavage

In order to map the F₁ subunit of the RS virus fusion protein, syntheticpolypeptides were prepared corresponding to various regions of the F₁subunit as depicted in FIG. 3 and designated synthetic polypeptides (sp)sp1 through sp5. These sp were coupled to KLH as described supra andemployed separately to immunize rabbits. Five corresponding rabbitantisera were obtained designated anti-sp1 through anti-sp5. All fiveantisera were reactive with the F₁ subunit as well as with theparticular sp used as immunogen.

The purified RS virus fusion protein was then subjected to definedproteolytic cleavage as described in detail Section 7.1.3. The cleavedprotein fragments were separated by SDS-PAGE to determine molecularweight and analyzed by Western blot analysis for ability to bind to theL4 monoclonal antibody as well as to the antisera raised against thesynthetic polypeptides. The position of a given proteolytic fragmentwithin the fusion protein sequence was deduced from the reactivitieswith each of the antisera. Results are tabulated in Table 11 (A-E) andsummarized in FIG. 4.

                  TABLE 11                                                        ______________________________________                                        MAPPING AND ANTIGENICITY OF PEPTIDE                                           FRAGMENTS OF F PROTEIN                                                        ______________________________________                                        A. Trypsin Digestion of F Protein                                             Fragment I        II       III    IV   V    VI                                Probe    28K-1    25K-1    24K    23K  15K  14K                               ______________________________________                                        L4       +        +        +      +    -    +                                 anti-sp (1)                                                                            +        +        +      +    +    -                                 anti-sp (4)                                                                            +        +        +      +    +    -                                 anti-sp (5)                                                                            -        -        +      -    -    -                                 Predicted                                                                              137-394  137-364  137-359                                                                              156- 137-                                   Amino Acid                        364  272                                    Position.sup.b                                                                ______________________________________                                        B. Trypsin Digestion of L4-Protected F Protein                                Fragment   VII           VIII   III                                           Probe      28K           26K    24K                                           ______________________________________                                        L4         +             -      +                                             anti-sp (1)                                                                              -             -      +                                             anti-sp (2)                                                                              +             +      -                                             anti-sp (4)                                                                              -             -      +                                             Predicted Amino                                                                          272-520       336-574                                                                              137-359                                       Acid Position.sup.d                                                           ______________________________________                                        C. Trypsin Digestion of L4-Protected F Protein Followed By                    Arg-C Digestion                                                               Fragment   III       IV       IX      V                                       Probe      24K       23K      20K     15K                                     ______________________________________                                        L4         +         +        +       -                                       anti-sp (4)                                                                              +         +        +       +                                       Predicted Amino                                                                          137-359   156-364  156-336 137-272                                 Acid Position.sup.c                                                           ______________________________________                                        D. Arg C Digestion of F Protein                                               Fragment   X        XI       XII    XIII  XIV                                 Probe      25K      23.5K    21K    14K   13K                                 ______________________________________                                        L4         +        -        +      +     +                                   anti-sp (2)                                                                              -        +        -      -     -                                   anti-sp (4)                                                                              -        -        +      -     -                                   anti-sp (5)                                                                              -        +        -      -     -                                   Predicted Amino                                                                          282-507  336-553  235-429                                          Acid Position                                                                 ______________________________________                                        E. Lys C Digestion of L4 Protected F Protein                                  Followed By Arg C Digestion                                                   Fragment X      XV     XI    XII  XVI  XVII  XVIII                            Probe    25K    25K    23.5K 21K  16K  14K   13K                              ______________________________________                                        L4       +      -      -     +    -    +     +                                anti-sp (2)                                                                            -      +      +     -    -    -     -                                anti-sp (4)                                                                            -      -      -     +    +    -     -                                anti-sp (5)                                                                            -      -      -     -    -    -     -                                Predicted                                                                              282-   327-   156-  235- 137- 272-  282-                             Amino Acid                                                                             507    553    336   429  282  399   399                              Position                                                                      ______________________________________                                         .sup.a The Roman Numerals identify the fragments shown in FIG. 4.             Approximate molecular weight (daltons) of cleavage fragments are indicate     under Roman Numerals.                                                         .sup.b The predicted amino acid positions of the various fragments were       deduced from their reactivity with the antisynthetic polypeptide sera as      well as the known amino acid specificities of the proteases.             

Table 11 (A-E) shows the fragments which were derived by the fiveproteolytic cleavage protocols along with the reactivity to themonoclonal and polyclonal antisera. FIG. 4 consolidates the data ofTable 11 (A-E), and shows the position of all of the fragmentsidentified in a linear map. The fragments which reacted with the L4monoclonal antibody are show as shaded bars and those which did notreact are shown in sold bars. The cross-hatched area present in allfragments reactive with L4 monoclonal antibody spans amino acid residues283-327.

8.3. Mapping by Expression of Protein Fragments

The cDNA substantially as depicted in FIG. 1 containing the completenucleotide sequence of the fusion protein gene was cloned into E. coliplasmid vector pG103 at the BamHI site. FIG. 5 illustrates the resultantrecombinant plasmid pPX1043.

The cDNA substantially as depicted in FIG. 1 containing the completenucleotide sequence of the fusion protein gene was cloned into E. coliplasmid vector pBR322 at the BamHI site. The resultant plasmid wasdesignated pBR322-F. Regions of the fusion protein gene were excisedfrom pBR322-F by restriction endonuclease digestion and ligated into theE. coli expression vector pUC19 (Yanish-Perron et al., 1985, Gene33:103-19). Table 12 shows the restriction sites used, the nucleotidesequences of the fusion protein gene within the restriction fragmentsand the amio acids encoded by these nucleotide sequences. Each of theseDNA fragments was cloned into the E. coli expression vector pUC19 in thepolylinker region such that the sequences were in frame with the lac Zgene initiation codon. The amino acid sequences encoded by therecombinant DNA molecules at and near the junctions of the vectorplasmid and the cloned fusion protein gene fragments are shown in FIG.6. Since coding sequences of the fusion protein were inserted into thelac Z gene of the pUC19 vector, some amino acids from the lac Z proteinas well as some amino acids encoded by the polylinker are included inthe recombinant proteins.

                  TABLE 12                                                        ______________________________________                                        FRAGMENTS OF FUSION PROTEIN GENE CLONED                                       INTO THE pUC19 EXPRESSION VECTOR                                                     Re-                                                                    Con-   combinant Restriction                                                                             Nucleotide                                                                             Amino Acid                                struction                                                                            Protein   Sites Used.sup.a                                                                        Boundaries.sup.b                                                                       Boundaries                                ______________________________________                                        cF3    F3        HpaI-BamHI                                                                              769-1737 253-574                                   cF4    F4        PstI-NsiI 646-1479 212-490                                   cF7    F7        PstI-TaqI 646-1102 212-363                                   cF8    F8        PstI-HincII                                                                             646-1165 212-384                                   cF10   F10       PstI-NdeI 646-907  212-298                                   cF11   F11       NdeI-NscI 908-1479 299-490                                   ______________________________________                                         .sup.a A clone of the RSV fusion protein gene was cleaved with the            indicated combinations of restriction endonucleases to generate RSV           fragments for subsequent cloning into the expression vector pUC19. The        nucleotides included within these restriction fragments are indicated         along with the predicted amino acids which were expressed.                    .sup.b Nuclestide 1900 is the beginning of the synthetic BamHI linker.        Nucleotides 1742-44 is a translation termination signal (TAA) so that         amino acid 574 is a terminal amino acid.                                 

Six recombinant proteins, designated F3, F4, F7, F8, F10, and F11,corresponding to proteins encoded by construction cF3 (also designatedas plasmid pPX-1029), cF4, cF7, cF8, cF10 and cF11, were expressed in E.coli as demonstrated by reactivity to polyclonal rabbit antiserum to thenative fusion proteins by Western blot analysis as described supra. Allof the recombinant proteins except F11 reacted with the L4 monoclonalantibody by Western blot analysis. FIG. 7 shows a diagramatic scheme ofthe L4 reactive and L4 non-reactive recombinant proteins. As illustratedin FIG. 7, the RS virus fusion protein sequence common to all of therecombinant proteins which are reactive with the L4 monoclonal antibodyis defined by residues 253 to 298.

8.4. Mapping by Synthetic Peptides

Four synthetic polypeptides were prepared which corresponded to theamino acid residues 299-315, 294-315, 289-315, and 283-315 of the RSvirus fusion protein respectively. The exact sequences of these foursynthetic polypeptides are shown in Table 13.

                                      TABLE 13                                    __________________________________________________________________________    SEQUENCE OF SYNTHETIC POLYPEPTIDES TESTED FOR REACTIVITY                      WITH THE L4 MONOCLONAL ANTIBODY                                               Peptide                                                                            Sequence.sup.a            Reactivity with L4                             __________________________________________________________________________    299-315                                                                            YVVQLPLYGVIDTPCWK         -                                              294-315                                                                            EEVLAYVVQLPLYGVIDTPCWK    +                                              289-315                                                                            MSIIKEEVLAYVVQLPLYGVIDTPCWK                                                                             +                                              283-315                                                                            QQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWK                                                                       +                                              __________________________________________________________________________     .sup.a Sequences are given by the standard single letter amino acid code.

The polypeptides were spotted on nitrocellulose sheets and tested forreactivity with the L4 monoclonal antibody by dot blot analysis (seeSection 7, supra).

Results obtained are illustrated in FIG. 8. As demonstrated in FIG. 8,peptide 299-315 did not react with the L4 monoclonal antibody whereaspolypeptides 294-315, 289-315 and 283-315 did, indicating that aneutralizing and fusion epitope resides between residues 283-298 and canbe as small as 294-299. The reasons why the minimum region is set at294-299 rather than 294-298 is because generally a stretch of at leastsix amino acids is necessary to form an epitope.

9. Immunogenicity of Modified RS Virus Fusion Protein 9.1.Conformational Modification

Crosslinking experiments demonstrated that the RS virus fusion proteinexists as a dimer (140,000 daltons) on the surface of virus infectedcells and in substantially pure preparations. In order to investigatethe potential effects of protein conformation on the vaccine utility,the quartenary structure of the F protein was modified and resultanteffects on immunogenicity evaluated.

A monomeric F protein (70,000 daltons) was obtained from substantiallypure F protein by treating the F protein at 100° C. for two minutes orby treatment with 99.9% acetonitrile /0.1% TFA. The immunogenicity ofdimeric and monomeric forms was tested in cotton rats. Experimentaldetails and results are presented in Table 14.

                  TABLE 14                                                        ______________________________________                                        IMMUNOGENICITY OF RSV-F PROTEIN:                                              EFFECT OF PROTEIN CONFORMATION.sup.a                                                    Dose & Route       EIA    Neut. Titer                               Immunogen*                                                                              ug (times)  N      log 10 log 10                                    ______________________________________                                        F-dimer   5 (3× IM)                                                                           15     5.7    3.8                                       F-monomer 5 (3× SC)                                                                            5     <2.0   <1.7                                      ______________________________________                                         .sup.a The monomeric form was derived from native dimeric form of the F       protein by heat treatment. After the treatments, cotton rats were             immunized (intramuscular or subcutaneous) 3 times with the indicated          immunogens at 2 week intervals and at week 6. Serum was collected and         assayed for their titers.                                                

The total neutralizing antibody titers were found to be 100-1000 foldless in the group that received the monomeric form, prepared by heattreatment.

9.2. Deacylated Fusion Protein

In order to determine the effect of covalently attached fatty acid onthe immunogenicity of RS virus, the fusion protein was deacylated usinghydroxylamine (1M for 4 hours at room temperature) and the efficacy ofthe deacylated protein was compared to that of F protein. Experimentaldetails and results are presented in Table 15.

                  TABLE 15                                                        ______________________________________                                        IMMUNOGENICITY AND PROTECTIVE EFFICACY OF F                                   PROTEIN: EFFECT OF FATTY ACID DEACYLATION.sup.a                               Immunogen                      RSV isolation and                              Type Amount          Pre-challenge GMT                                                                         titers from lungs.sup.b                      (ug)      N      EIA-F   Neut (A2)                                                                             (+)  (-)  GMT                                ______________________________________                                        F    0.5      5      156,000                                                                             233     5    0    3.36                                  5.0      3      163,000                                                                             429     2    1    2.04                             deacylated                                                                    F    0.5      6      170,000                                                                             231     5    1    3.31                                  5.0      6      363,000                                                                             510     6    0    3.20                             PBS  0.0      6      <100  <20     6    0    4.32                             ______________________________________                                         .sup.a Cotton rats were immunized with the indicated immunogens at week 0     2 and 4. At week 6, they were challenged with RSV/A2 virus intranasally.      Serum was collected from the day of challenge and assayed.                    .sup.b "+" represents the number of animals with detectable RS virus in       their lungs at 4 day postchallenge. "-" represents the number of animals      without detectable RS virus in their lungs at 4 days postchallenge. GMT       titers are expressed as PFU/GM tissue.                                   

Experimental results indicate that the protective efficacy of thedeacylated F protein at 5 ug dose was reduced compared to the acylated Fprotein.

10. Protection of Animals: RS Virus G Protein 10.1. Isolation of GProtein

Substantially pure RS virus G protein suitable for use as an immunogenin a subunit vaccine formulation was prepared essentially according tothe method of Walsh et al., 1984, J. Gen. Virol. 65:761-67.

10.2. Passive Protection

Rabbits were immunized using the affinity purified G protein with anintramuscular injection of 2.4 ug of RS virus G protein in Freund'sComplete Adjuvant at week 0 and with 4.8 ug of G protein in Freund'sIncomplete Adjuvant at weeks 4 and 8. Animals were exsanguinated on week17. The IgG fractions of sera from the G-immunized rabbits and from acontrol group of non-immunized rabbits were obtained using a protein ASepharose™ column.

Twenty cotton rats were divided into 3 groups of 4 animals each and twogroups of 5 and 3 animals each. The animals were passively immunized byintraperitoneal injection as follows: Group 1 received 4.12 mg of rabbitanti-G IgG; Group 2, 1.03 mg of rabbit anti-G IgG; Group 3, 4.28 mg ofnormal rabbit IgG; Group 4, 1.07 mg of normal rabbit IgG; and Group 5(control), an equivalent volume of PBS. Twenty-four hourspost-immunization, animals were challenged with 5.5 log10 PFU RS virusLong strain. Lungs were harvested on day 4 post-challenge. The presenceand/or quantity of RS virus in lung tissues was determined as describedabove. Serum samples were collected and assays were conducted asdescribed above. Results are presented in Table 16.

                  TABLE 16                                                        ______________________________________                                        PROTECTIVE EFFICACY OF PASSIVE                                                IMMUNIZATION OF ANTI-G ANTIBODIES                                                        Serology.sup.b                                                     Amount of IgG       Virus       Virus Assay.sup.c                             Administered.sup.a                                                                         EIA    Neutralization                                                                            +   -   TITER                                 ______________________________________                                        Anti G-IgG 4.12 mg                                                                         5.5    3.3         0   4   ≦1.0                           Anti G-IgG 1.03 mg                                                                         4.8    3.0         0   4   ≦1.0                           Normal IgG 4.28 mg                                                                         1.0    2.0         5   0   4.5                                   Normal IgG 1.07 mg                                                                         1.0    2.0         3   0   4.9                                   none (PBS)   1.0    2.0         4   0   5.0                                   ______________________________________                                         .sup.a Cotton rats were passively immunized using an intraperitoneal          injection of rabbit immunoglobulin elicited using purified G protein from     RS virus Long stain. The number of animals in each group was four, except     those which received 4.28 mg normal rabbit IgG (5 animals) and 1.07 mg        normal rabbit IgG (3 animals).                                                .sup.b Assays were performed on serum samples obtained on the day of          challenge (see text for details). Results represent the geometric mean        titer for 3-5 animals expressed in log.sub.10 units (EIA) and log.sub.10      units/ml serum (Virus Neutralization Assay).                                  .sup.c Long strain RS virus 5.5 log 10 PFU was inoculated intranasally        (IN) into the passively immunized animals at 24 hours postimmunization an     lungs were harested at 4 days postchallenge. Virus was isolated from the      lung tissues. "+" indicates the number of animals in each group in which      virus was detectable in lungs. "-" indicates the member in which no virus     was detected. "Titer" represents the geometric mean titer (PFU/gm of          tissue) of virus isolat ed from lung tissue.                             

As demonstrated in Table 16, passive administration of rabbit antibodyinduced by purified G protein from RS virus was efficacious inprotecting cotton rats against infection by homologous RS virus.

10.3. Active Immunization and Protection

Thirty seven cotton rats were divided into 3 groups of 12-13 animalseach. One group was then further subdivided into 4 groups of 3 animalseach, thus forming 6 groups. All animals in all groups, except Group 6,were immunized by intramuscular injection of 10 ug of RS virus Gprotein, obtained as described in Section above, in different adjuvantsas follows: Group 1 received three injections at weeks 0, 2 and 4-5 of Gprotein in PBS; Group 2, three injections at weeks 0, 2 and 5 of Gprotein in Alum; Group 3, two injections at weeks 0 and 5 of G proteinin Alum; Group 4, three injections at weeks 0, 2 and 5 of G protein inISCOM; and Group 5, two injections at weeks 0 and 5 of G protein inISCOM. Group 6, the control group, received three injections at weeks 0,2 and 4 of an equivalent volume of PBS alone. Between weeks 6 and 7, allanimals were challenged intranasally with 4.0-5.0 log 10 PFU RS virusLong strain. Serum samples were collected on the day of challenge andassayed as described above in Section 6.2, supra. The presence and/orquantity of RS virus in lung tissues was determined at 4 dayspost-challenge as described above. Results are presented in Table 17.

                  TABLE 17                                                        ______________________________________                                        IMMUNOGENICITY AND PROTECTIVE EFFICACY OF                                     RS VIRUS G PROTEIN                                                                  Immunogen          Neu-  Serology.sup.b                                 Group (Adju-             tra-  Virus Assay.sup.c                              No.   vant).sup.a                                                                             N     EIA  lization                                                                            N    +    -   GMT                            ______________________________________                                        1     (PBS)     12     3.4 4.0   6    1    5    1.4                           2     (Alum)     3     6.1 5.9   3    0    3   <1.0                           3     (Alum)     3     6.3 5.3   3    0    3   <1.0                           4     (ISCOM)    3     6.4 5.7   3    0    3   <1.0                           5     (ISCOM)    3     6.3 5.3   3    0    3   <1.0                           6     PBS       13    <1.0 <2.0  13   13   0    4.3                           ______________________________________                                         .sup.a In all cases, the immunogen was 10 ug G protein in an adjuvant as      indicated.                                                                    .sup.b Assays were performed on serum samples obtained on the day of          challenge. Results represent the geometric mean titer expressed in            log.sub.10 units.                                                             .sup.c Virus was isolated from the lung tissues. "N" indicates the number     of animals examined. "+" indicates the number of animals in each group in     which virus was detectable. "-" indicates the member in which no virus wa     detected. "Titer" represents the geometric mean titer (PFU/gm of tissue)      of virus isolated from lung tissue.                                      

As illustrated in Table 17, active immunization of cotton rats with Gprotein obtained from RS virus Long strain induced a significant immuneresponse as demonstrated by high titers of anti-G antibody (EIA assay)that were capable of neutralizing RS virus Long strain (Neutralizationassay). Most importantly, when immunized animals were challenged with RVvirus Long strain, they were effectively protected against suchinfection as compared to control animals.

11. IMMUNOGENICITY OF RS VIRUS G PROTEIN EXPRESSED IN RECOMBINANTVECTORS

A recombinant vector expressing the RS virus Strain A2 G protein wasprepared. Plasmid pPL-lambda (pPL), which carries the P_(L) promoter andthe N gene on a 1215 bp segment of the bacgeriophage lambda genome waspurchased from Pharmacia Fine Chemicals (Piscataway, NJ). A unique HpaIsite in the N gene is located 321 bp downstream from the start of P_(L)transcription. Sequences inserted into this restriction site will thisbe regulated by the P_(L) promoter. An expression vector plasmid pPX1600was constructed by inserting a synthetic ologinucleotide sequence intothe HpaI site of the pPL plasmid. This oligonucleotide contained atranslation termination codon in frame with the N coding sequencesfollowed by translation initiation signals (Shine-Dalgarno box plus ATG)and unique restriction sites (NcoI, StuI, EcoRV) for insertion ofheterologous DNA sequences in frame with the synthetic ATG. A fulllength cDNA corresponding to the G glycoprotein gene that was flanked byBamHI linkers (Elango et al., 1980, Proc. Nat'l Acad. Sci. USA83:1906-10) was cloned into the BamHI site of pBR322. After BamHIdigestion, the RS virus gene encoding the G protein was excised frompBR322, filled with the Klenow fragments of DNA polymerase I, and bluntend ligated to StuI-cleaved pPX1600. Following transformation of E. coli(N99cI⁺ strain), the plasmid pPX1044 was isolated (FIG. 9). In thisplasmid, the RS virus G encoding sequence was in the correct orientationand reading frame for P_(L) -directed expression by readthrough from thesynthetic ATG.

Plasmid pPX1044 was introduced into Salmonella typhimurium (LB50150)cells by the CaCl₂ procedure. The transformed cells were grown to latelog phase and the expressed RS virus G protein was purified by animmunoaffinity method using L9 monoclonal antibody (Walsh and Hruska,1983 J. Virol. 47:171-177). The recombinant non-glycosylated G proteinproduced significant neutralization titers (3.7 logs/ml against Longsrain) in rabbits, following 4 immunizations with 10 ug in the presenceof Freund's adjuvant. A similar neutralizing antibody response waselicited in cotton rats after 3 immunizations with 5ug of therecombinant non-glycosylated G protein. Hence, the bacterially derivedrecombinant G is immunogenic and the antibodies produced against therecombinant G are functional and therefore will be protective against RSvirus infection.

12. CELL-MEDIATED IMMUNOLOGICAL ASPECTS OF RS VIRUS VACCINE

The data presented in this application demonstrate that immunizationwith RSV glycoprotein(s) either F protein alone or in combination with Gprotein, induces circulating antibodies that are functional andprotective. In addition to this antibody response, cell mediatedimmunity may be of importance for the protective efficacy. Indeed,several studies have shown that many viral infections are associatedwith the induction of cytotoxic T cells which recognize viralglycoproteins. Cytotoxic T cells against RS virus have been described inmouse and humans (Taylor, et al., 1984, Infect. Immun. 43:649-55;Bangham et al., 1985, J. Virol 56:55-59; Bangham et al., 1986, Proc.Nat'l Acad Sci. USA 83:9183-87). Moreover, it has been shown thatpassive transfer of T cells from RSV-primed mice can clear a presistentRSV infection when the cells are administered to athymic nude mice(Cannon et al., 1987, Immunol. 62:133-38). In order to investigatewhether immunization with RS virus F protein could induce cytotoxic Tcell responses, mice were immunized with purified RS virus F protein andimmune-stimulated effector T cells were assayed in vitro. Responses inanimals immunized with live RS virus were also examined. In practice, 36mice were divided into 4 groups and animals were immunized at 0 and 2months as follows: Group 1 received 2×10⁶ TCID50 Long strain RS virusintranasal and intraperitoneal injections respectively; Group 2, 20 ugRS virus F protein (in Alum) via intranasal injections; Group 3, 20 ugRS virus F protein (in Alum) via intramuscular injections; and Group 4,were a non-immunized control group. Three week after the last boosterinjection, animals were sacrificed and spleens were harvested. EffectorT cells were obtained from the spleens and were re-stimulated in vitrowith either peritoneal exudate cells (PEC) or spleen cells infected withRS virus. Cytotoxic T cells were tested using a Cr-51 release assay(Bangham et al., 1985, J. Virol. 56:55-59). The results are presented inTable 18.

                  TABLE 18                                                        ______________________________________                                        CTL RESPONSE INDUCED BY RSV-F PROTEIN                                         In Vitro   % Cr Release From Target Cellsb                                    Group Stimu-   RSV + PEC  BCH-4    RSV - PEC.sup.c                            No..sup.a                                                                           lation   40:1   10:1  40:1  10:1 40:1  10:1                             ______________________________________                                        1     RSV +     10.1  3.9   6.2   -4.2 0.8   0.2                                    PEC                                                                           RSV +     23.8  10.0  15.2  -2.0 -0.2  1.1                                    spleen                                                                        None     -0.2   --    -0.2  --   -0.3  --                               2     RSV +     12.4  4.0   9.2   -2.6  0.97 -0.8                                   PEC                                                                           RSV +    -0.2   -0.2  -1.4  -0.6 -0.1  1.5                                    spleen                                                                        None     -0.7   --    1.6   --   -0.8  --                               3     RSV +     2.4   2.4   8.4    1.2 0.4   -0.8                                   PEC                                                                           RSV +    -0.3   -0.1  -6.0  - 5.3                                                                              0.8   -1.6                                   spleen                                                                        None     -0.2   --    0.2   --   -5.8  --                               4     RSV +     0.4   --    5.8   --   0.5   --                                     PEC                                                                           RSV +    -0.4   --    1.0   --   -0.2  --                                     spleen                                                                        None     -1.5   --    0.6   --   0.2   --                               ______________________________________                                         .sup.a See text for experimental details.                                     .sup.b Target cells were BCH.sub.4 cells (a persistently RSV Long strain      infected Balb/c fibroblast cell line), RSV infected peritoneal exudate        cells (PEC) and uninfected PEC cells.                                         .sup.c The ratio indicates the ratio of effector: target cells.          

Significant levels of RS virus specific cytotoxic T cells were inducedfollowing immunization with RS virus F protein (IP/IP) i.e. Group 2 andwith RS virus (IN/IP), i.e., Group 1. No significant T cell response wasobserved in animals which received RS virus F protein via anintramuscular route, i.e., Group 3 or in control animals, i.e., Group 4.The induction of cytotoxic T cells thus depends upon the route ofinoculation, proteins versus live virus and the cells used forrestimulation. Purified F protein of RSV is able to induce not onlyhumoral but also cellular immunity.

13. DEPOSIT OF MICROORGANISMS

Many polynucleotide sequences may be used to practice the presentinvention. E. coli strain JM83 carrying plasmid pPX1043 which comprisesthe complete RS Virus fusion protein gene and an E. coli strain JM103carrying plasmid pPX1029 have been deposited with the AgriculturalResearch Culture Collection (NRRL) Peoria, IL and have been assignedaccession numbers NRRL B-18254 and NRRL B-18255 respectively. E. colistrain N99cI⁺ carrying plasmid pPX1044 has also been deposited with theAgricultural Research Culture Collection (NRRL), Peoria, Ill. and hasbeen assigned accession number B-18419.

The present invention is not to be limited in scope by themicroorganisms deposited, since the deposit of microorganisms is onlyintended as a single illustration of one aspect of the invention.

Many variations of this invention as herein set forth may be madewithout departing from the spirit and scope thereof. Such variations areintended to fall within the scope of the appended claims. The specificembodiments described are given by way of example only, and theinvention is limited only by the appended claims.

What is claimed is:
 1. A method for protecting a primate from a diseaseinduced by Respiratory Syncytial virus, comprising: administering to theprimate an effective amount of a vaccine formulation which comprises asubstantially pure polypeptide which is a human Respiratory Syncytialvirus fusion protein, characterized by purity of greater than 75% byweight, a molecular weight of about 140,000 daltons, and having a nativedimeric form, in combination with an adjuvant suitable foradministration to the primate.
 2. A method for protecting a human fromdisease induced by Respiratory Syncytial virus, comprising:administering to the human an effective amount of a vaccine formulationwhich comprises a substantially pure polypeptide which is a humanRespiratory Syncytial virus fusion protein, characterized by purity ofgreater than 75% by weight, a molecular weight of about 140,000 daltons,and having a native dimeric form, in combination with an adjuvantsuitable for administration to the human.
 3. The method according toclaim 2, in which the adjuvant is alum.